The Sasanian period marks the end of the ancient and the beginning of the medieval era in the history of the Middle East. Universalist religions such as Christianity, Manichaeism, and even Zoroastrianism and Judaism absorbed local religions and cults at the beginning of the 3rd century. Both the Sasanian and the Roman empires ended by adopting an official state religion, Zoroastrianism for the former and Christianity for the latter. In Mesopotamia, however, older cults such as that of the Mandaeans, the moon cult of Harran, and others continued alongside the great religions. The new rulers were not as tolerant as the Seleucids and Parthians had been, and persecutions occurred under Sasanian rule.

After Ardashir I, the first of the Sasanians, consolidated his position in Persis (modern Fars province), he moved into southern Mesopotamia, and Mesene submitted. In 224 he defeated and killed the last Parthian ruler, Artabanus V, after which Mesopotamia quickly fell before him and Ctesiphon became the main capital of the Sasanian empire. In 230 Ardashir besieged Hatra but failed to take it. Hatra called on Roman aid, and in 232 the Roman emperor Severus Alexander launched a campaign that halted Ardashir's progress. At the death of Severus Alexander in 235 the Sasanians took the offensive, and probably in 238 Nisibis and Harran came under their control. Hatra was probably captured in early 240, after which Ardashir's son Shapur was made coregent; Ardashir himself died soon afterward. The Roman emperor Gordian III led a large army against Shapur I in 243. The Romans retook Harran and Nisibis and defeated the Sasanians at a battle near Resaina, but at Anbar, renamed Peroz-Shapur ("Victorious Is Shapur"), the Sasanians inflicted a defeat on the Romans, who lost their emperor. His successor, Philip the Arabian, made peace, giving up Roman conquests in northern Mesopotamia. Osroene, however, which had been returned to the local ruling family of Abgar by Gordian, remained a vassal state of the Romans. Shapur renewed his attacks and took many towns, including Dura-Europus, in 256 and later moved into northern Syria and Anatolia. The defeat and capture of the Roman emperor Valerian at the gates of Edessa, probably in 259, was the high point of his conquests in the west. On Shapur's return to Ctesiphon the ruler of Palmyra, Septimius Odaenathus (also called Odainath), attacked and defeated his army, seizing booty. Odeanathus took the title of emperor, conquered Harran and Nisibis, and threatened Ctesiphon in 264-266. His murder relieved the Sasanians, and in 273 the Roman emperor Aurelian sacked Palmyra and restored Roman authority in northern Mesopotamia. Peace between the two empires lasted until 283, when the Roman emperor Carus invaded Mesopotamia and advanced on Ctesiphon, but the Roman army was forced to withdraw after Carus' sudden death. In 296 Narseh I, the seventh Sasanian king, took the field and defeated a Roman force near Harran, but in the following year he was defeated and his family was taken captive. As a result, the Romans secured Nisibis and made it their strongest fortress against the Sasanians. The Roman province of Mesopotamia, which was the land between the Euphrates and Tigris in the northern foothills, became in effect a military area with limes (the fortified frontiers of the Roman Empire) and highly fortified towns.

Under Shapur II the Sasanians again took the offensive, and the first war lasted from 337 to 350; it ended with no result as Nisibis was successfully defended by the Romans. In 359 Shapur again invaded Roman territory and captured the Roman fortress Amida after a long and costly siege. In 363 the emperor Julian advanced almost to Ctesiphon, where he died, and his successor Jovian had to give up Nisibis and other territories in the north to the Sasanians. The next war lasted from 502 to 506 and ended with no change. War broke out again in 527, lasting until 531, and even the Byzantine general Belisarius was not able to prevail; as usual, the boundaries remained unchanged. In 540 the Sasanian king Khosrow (Chosroes) I invaded Syria and even took Antioch, although many fortresses behind him in northern Mesopotamia remained in Byzantine hands. After much back-and-forth fighting, peace was made in 562. War with the Byzantine Empire resumed 10 years later, and it continued under Khosrow's successor, Hormizd IV. Only in 591, in return for their assistance in the restoration to the Sasanian throne of Khosrow II, who had been deposed and had fled to Byzantine territory, did the Byzantines regain territory in northern Mesopotamia. With the murder in 602 of the Byzantine emperor Maurice, who had been Khosrow's benefactor, and the usurpation of Phocas, Khosrow II saw a golden opportunity to enlarge Sasanian domains and to take revenge for Maurice. Persian armies took all northern Mesopotamia, Syria, Palestine, Egypt, and Anatolia. By 615, Sasanian forces were in Chalcedon, opposite Constantinople. The situation changed completely with the new Byzantine emperor Heraclius, who, in a daring expedition into the heart of enemy territory in 623-624, defeated the Sasanians in Media. In 627-628 he advanced toward Ctesiphon, but, after sacking the royal palaces at Dastagird, northeast of Ctesiphon, he retreated.

After the death of Khosrow II, Mesopotamia was devastated not only by the fighting but also by the flooding of the Tigris and Euphrates, by a widespread plague, and by the swift succession of Sasanian rulers, which caused chaos. Finally in 632 order was restored by the last king, Yazdegerd III, but in the following year the expansion of the Muslim Arabs began and the end of the Sasanian empire followed a few years afterward.

Unlike the Parthians, the Sasanians established their own princes as rulers of the small kingdoms they conquered, except on the frontiers, where they accepted vassals or allies because their hold over the frontier regions was insecure. By placing Sasanian princes over the various parts of the empire, the Sasanians maintained more control than the Parthians had. The provincial divisions were more systematized, and there was a hierarchy of four units--the satrapy (shahr in Middle Persian), under which came the province (ostan), then a district (tassug), and finally the village (deh). In Mesopotamia these divisions were changed throughout Sasanian history, frequently because of Roman invasions.

Many native tax collectors were replaced by Persians, who were more trusted by the rulers. In addition to the many tolls and tariffs, corvée, and the like, the two basic taxes were the land and poll taxes. The latter were not paid by the nobility, soldiers, civil servants, and the priests of the Zoroastrian religion. The land tax was a percentage of the harvest, but it was determined before the collection of the crops, which naturally caused many problems. Khosrow I undertook a new survey of the land and imposed the tax in a prearranged sum based on the amount of cultivable land, the quantity of date palms and olive trees, and the number of people working on the land. Taxes were to be paid three times a year. Abuses were still rampant, but this was better than the old system; at least, if a drought or some other calamity occurred, taxes could be reduced or remitted. Although information is contradictory, it appears that religious communities other than the Zoroastrian one had extra taxes imposed on them from time to time. This was especially true of the growing Christian community, particularly in the time of Shapur II, after Christianity became the official religion of the Roman Empire.

Religious communities became fixed under the Sasanians, and Mesopotamia with its large Jewish and Christian populations experienced changes because of the shift in primary allegiance from the ruler to the head of the religious group. The exilarch of the Jews had legal and tax-collecting authority over the Jews of the Sasanian empire. Mani, the founder of the Manichaean religion, was born in lower Mesopotamia, and his religion spread quickly both to the east and west, even before his death. In its homeland, Mesopotamia, it came under severe persecution by the priests of the Zoroastrian religion, who viewed Manichaeism as a dangerous heresy. Christianity, however, was viewed not as a heresy but as a separate religion, tolerated until it became the official religion of the enemy Roman Empire; Christians were then regarded as potential traitors to the Sasanian state. The first large growth of Christianity in Mesopotamia came with the deportation and resettlement of Christians, especially from Antioch with its patriarch, during Shapur I's wars with the Romans. In a synod convened in 325, the metropolitan see of Ctesiphon was made supreme over other sees in the Sasanian empire, and the first patriarch or catholicos was Papa. In 344 the first persecutions of Christians began; they lasted with varying degrees of severity until 422, when a treaty with the government ended the persecutions.

The earliest contemporary mention of Christians in Mesopotamia is in the inscriptions of Karter, the chief Zoroastrian priest after the reign of Shapur I. He mentions both Christians and Nazareans, possibly two kinds of Christians, Greek-speaking and Syriac-speaking, or two sects. It is not known which groups are meant, but it is known that followers of the Gnostic Christian leaders Bardesanes (Bar Daisan) and Marcion were active in Mesopotamia. Later, after the Nestorian church separated from the Monophysites, whose centre was in Antioch, the Nestorian church dominated Mesopotamia until the end of the Sasanian dynasty, when the Monophysites were growing in numbers. After about 485 the Sasanian government was satisfied that the Nestorian church in their domains was not loyal to Byzantium, and further persecutions were not state-inspired but rather prosecuted by the Zoroastrian clergy. At the end of the Sasanian period, the Nestorians were fighting the Monophysites, now called Jacobites, more than the Zoroastrians. The Jacobites established many monasteries, especially in northern Mesopotamia, whereas the Nestorians were cool toward monasticism.

Ethnicity became less important than religious affiliation under the Sasanians, who thus changed the social structure of Mesopotamia. The Arabs continued to grow in numbers, both as nomads and as settled folk, and Arabic became widely spoken. King Nu'man III of the Arab client kingdom of the Lakhmids of Al-Hirah in southern Mesopotamia became a Christian in 580, but in 602 he was deposed by Khosrow II, who made the kingdom a province of the empire. This act removed a barrier against inroads by Arab tribesmen from the desert, and, after the union of Arabs in the peninsula under the banner of Islam, the fate of the Sasanian empire was sealed. The Muslims, on the whole, were welcomed in Mesopotamia as deliverers from the foreign yoke of the Persians, but the conversion of the mass of the population to Islam did not proceed rapidly, mainly because of the well-organized Christian and Jewish communities. The arrival of Islam, of course, changed the history of Mesopotamia more than any other event in its history.