In this study, samples taken from the remaining parts of the city wall of the Old City of Herat were analyzed through radiocarbon dating to determine the date of construction for the first time. The results indicate that the city wall was most probably constructed between AD 441-649 (likely between AD 535-649). AD 535-649 roughly coincides with the period between the construction of the defensive wall of Gorgan and the destruction of the Sasanian dynasty by the Arab-Islamic powers. The hypothesis is that Khosrow I built the city wall as a fortified settlement at the end of the sixth century.