In the year 333 BC , Alexander the Great, the king of Macedonia defeated the Persian king Darius III near the village of Issus ( near Iskenderun) and undertook the control of the eastern Mediterranean as well as the continental trade routes. After Alexander's death (323 BC) his generals divided up the territory he had conquered, and Seleucus I Nicator, founder of the Seleucid dynasty, received the large part of Alexander's divided empire. Defeating Antigonus, another of Alexander's generals, at the battle of Ipsus in 301 BC Seleucus added Syria and the eastern part of Asia Minor to his domain. He founded Antioch in 300 BC and named the city after his father Antiochus (one of many in the Seleuced family to bear that name). The city was built on the eastern banks of the Orontes River (Asi River) and was known as Antioch on the Orontes to distinguish it from Antioch of Pisidia (located in Yalvaç in the province of Isparta) which was also established by Seleucus I and named for his father.

Antioch served as the capital of Seleucid empire for two centuries until the end of the Seleucid dynasty in about 83 BC During that time the city prospered, the population increased and owing to its mild climate, fertile farmlands and favorable location on one of the most important trade routes between Asia Minor and the lands to the south,became a commercial rival of Alexandria in Egypt. One of the most remarkable members of the Seleucid dynasty was Antiochus IV Epiphanes (175-163 BC). He expanded and beautified the city; added a new quarter to the city named Epiphania with an additional marketplace. The new quarter was needed to provide for an increase in the population. A Temple of Jupiter was constructed, which "had not only its ceiling panelled with gold, but also its walls wholly covered with gilded plates". One of the Antiochus IV's monuments, the rock hewn bust traditionally called the Charonion, is still visible on the mountainside above the city.

After the death of Antiochus IV in 163 BC, Seleucid Dynasty ceased to be a world power and started to decline. The city was occupied by the Armenian king Tigranes II between 83 - 69 BC. And then came the Romans in 64 BC.