Artaxerxes III was succeeded by Artaxerxes IV Arses, who before he could act was also poisoned by Bagoas. Bagoas is further said to have killed not only all Arses' children, but many of the other princes of the land. Bagoas then placed Darius III, a nephew of Artaxerxes IV, on the throne. Darius III, previously Satrap of Armenia, personally forced Bagoas to swallow poison. In 334 BCE, when Darius was just succeeding in subduing Egypt again, Alexander and his battle-hardened troops invaded Asia Minor.

Alexander the Great (Alexander III of Macedon) defeated the Persian armies at Granicus (334 BCE), followed by Issus (333 BCE), and lastly at Gaugamela (331 BCE). Afterwards, he marched on Susa and Persepolis which surrendered in early 330 BCE. From Persepolis, Alexander headed north to Pasargadae, where he visited the tomb of Cyrus, the burial of the man whom he had heard of from the Cyropedia.

Darius III was taken prisoner by Bessus, his Bactrian satrap and kinsman. As Alexander approached, Bessus had his men murder Darius III and then declared himself Darius' successor, as Artaxerxes V, before retreating into Central Asia leaving Darius' body in the road to delay Alexander, who brought it to Persepolis for an honourable funeral. Bessus would then create a coalition of his forces, in order to create an army to defend against Alexander. Before Bessus could fully unite with his confederates at the eastern part of the empire, Alexander, fearing the danger of Bessus gaining control, found him, put him on trial in a Persian court under his control, and ordered his execution in a "cruel and barbarous manner."

Alexander generally kept the original Achaemenid administrative structure, leading some scholars to dub him as "the last of the Achaemenids". Upon Alexander's death in 323 BCE, his empire was divided among his generals, the Diadochi, resulting in a number of smaller states. The largest of these, which held sway over the Iranian plateau, was the Seleucid Empire, ruled by Alexander's general Seleucus I Nicator. Native Iranian rule would be restored by the Parthians of northeastern Iran over the course of the 2nd century BCE.