Queen Gorgo of Sparta tells her men about the Battle of Marathon, in which King Darius of Persia was killed by General Themistocles of Athens ten years earlier. Darius's son, Xerxes, witnesses his father's death and is told "only the gods can defeat the Greeks". Darius's naval commander, Artemisia, claims that Darius' last words were a challenge, and sends Xerxes on a journey through the desert. Xerxes reaches a cave and bathes in an otherworldly liquid, emerging as an 8-foot tall "God-King". He returns to Persia and declares war on Greece to avenge his father.

As Xerxes's forces advance towards Thermopylae, Themistocles meets with the council and convinces them to provide him with a fleet to engage the Persians at sea. Themistocles travels to Sparta to ask King Leonidas for help, but is informed by Dilios that Leonidas is consulting the Oracle, and Gorgo is reluctant to side with Athens. Themistocles reunites with his old friend Scyllias, who infiltrated the Persian troops and reveals Artemisia was born Greek, but defected to Persia after her family was raped and murdered by Greek hoplites. Rescued and adopted by a Persian emissary, she was trained by the best Persian warriors, and eventually gained the attention of King Darius. Themistocles also learns that Leonidas has marched to fight the Persians with only 300 men.

Themistocles leads his fleet of fifty warships and several thousand men, which include Scyllias, Scyllias's son Calisto, and Themistocles' right-hand man Aeskylos to the Aegean Sea, starting the Battle of Artemisium. They ram their ships into the Persian ships and charge them, slaughtering several soldiers before retreating. The following day, the Greeks feign a retreat and lead a group of Persian ships into a crevice, where they become stuck. The Greeks charge the Persian ships from the cliffs above and kill more Persians. Impressed with Themistocles' skills, Artemisia brings him onto her ship and attempts to lure him to the Persian side as her second-in-command. They fight and make love simultaneously, and he refuses her offer.

The Persians attack the Greek ships with tar and flame bombs, but an Athenian kills one of the Persians, who falls into the tar carrying a torch, damaging ships from both sides. Themistocles is thrown into the sea by an explosion and nearly drowns before being rescued by Aeskylos, and stands by Scyllias's side as he succumbs to his injuries. Believing Themistocles to be dead, Artemisia and her forces withdraw. After recovering from his injuries, Themistocles learns that only a few hundred of his warriors and six of his ships survived the disastrous attack.

Daxos, an Arcadian general, tells Themistocles that Leonidas and his 300 men were killed after Ephialtes betrayed the Greeks to Xerxes. Themistocles returns to Athens and confronts Ephialtes, who reveals that Xerxes plans to attack and burn Athens to the ground. Ephialtes regrets his betrayal and welcomes death, but is spared so he can warn Xerxes that the Greek forces are gathering at Salamis. Themistocles visits Gorgo in Sparta to ask for help, but Gorgo is mourning Leonidas's death and angry for what the goal of a united Greece has cost. Before departing, Themistocles returns Leonidas's sword, which had been delivered to him by Ephialtes under Xerxes's orders, and urges Gorgo to avenge Leonidas.