He was the son of Molpagoras, and son-in-law of Histiaeus, whom the Persians had set up as tyrant of Miletus. Aristagoras gained control of the city when Histiaeus was appointed as an advisor to the Persian king Darius I. When Naxos revolted in 502 BC, the Persian overlords of the island asked Aristagoras for help, and he agreed in the assumption that he would be recognized as ruler of the island. He allied with Artaphernes, the Persian satrap of Lydia, and was given a fleet of ships. Unfortunately for the invasion, Aristagoras quarrelled with the admiral Megabates, who then informed the Naxians that the fleet was coming.
The invasion failed and the alliance with Artaphernes fell apart. In an attempt to save himself from the wrath of Persia he began to plan a revolt with the Milesians and the other Ionians. Meanwhile, Histiaeus, from his post in Persia, hoped to begin his own revolt in Miletus, so that the Persians would crush it and reinstall him as tyrant. In Miletus in 499 BC, Aristagoras was supported by most of the citizens, except the historian Hecataeus. A democracy was established, and soon the other Ionian cities had also revolted against the Persians, thus beginning the Ionian Revolt.
Aristagoras then travelled to mainland Greece to find support for the revolt. In Sparta he met with the king Cleomenes I, and argued that a pre-emptive invasion of Persia would be easy; there were many riches along the way, and the capital of Susa was "only" three months away. Cleomenes rejected his offer, but Aristagoras found more success in Athens, because, according to Herodotus, it was easier to convince an assembly of thousands of Athenians than it was to convince one Spartan king.
With Athenian help Aristagoras led the attack on Sardis, the Persian capital in Ionia. However, when the Ionian Revolt was eventually put down (and Histiaeus restored as tyrant), Aristagoras fled to Thrace, where he attempted to establish a colony on the Strymon river, at the same site as the later Athenian colony of Amphipolis. He was killed by the Thracians while attacking a neighbouring Thracian town.