Leonidas, king of Sparta, leads a small force of three hundred men—his personal bodyguard—to a place known as the Hot Gates to meet a numberless horde of hostile Persians in combat. Prevented by oracular pronouncement from taking the entire Spartan army, Leonidas is fully aware that the mission is suicidal. Joined by about 6,700 other warriors from various Greek cities, Leonidas and his three hundred hold off several assaults by the Persians until eventually they are betrayed.

As a young boy of perhaps ten years, Leonidas, like all Spartan boys, had been driven into the wilderness, alone and unarmed, to test his strength and cunning. Tracked by a huge wolf, Leonidas crafted a flimsy spear and lured the wolf into a narrow fissure in a cliff. The wolf pursued Leonidas until its movement was severely restricted whereupon the boy killed the wolf, returning victorious to Sparta where he was eventually to become king. After reigning for many years Leonidas receives the envoy of king Xerxes of Persia. The envoy demands a token gesture of subjugation to Xerxes—Leonidas refuses to be so abased and kills the envoy and his retinue.

The following year news of a massive Persian invasion reaches Sparta. Leonidas desires to sally against the Persians with the entire Spartan army but, as required by law, first seeks the advice and consent of the ephors—the elders of Sparta. In turn the ephors consult the mystic oracle, a young girl, who demands the Carneia, a religious festival, be observed and insists that no Spartan army mobilize during the religious period. Leonidas, stymied, returns to Sparta without realizing the Persians have successfully bribed the corrupt ephors. Prevented from mobilizing the Spartan army, Leonidas determines to take a walk through the countryside. The queen, sensing his motive, insists his entire personal bodyguard, numbering three hundred, accompany him. Leonidas subsequently walks north with his retinue until meeting—supposedly by happenstance—other soldiers assembling for the defense of Greece.

Leonidas, leading a mixed force of about 7,000 men, reaches a restrictive pass known variously as Hell's Mouth or the Hot Gates; history remembers the location as Thermopylae. There he readies his meager force to meet the vast Persian army, rumored to be composed of hundreds of nations and hundreds of thousands of men. A misshapen hunchback named Ephialtes approaches Leonidas in camp. Ephialtes explains that his father—a Spartan—had, after the birth of a deformed child, gone into exile rather than commit infanticide as required by Spartan law. Ephialtes presents himself as trained and capable, and states a desire to fight alongside the Spartans to regain the honor of his family. Leonidas declines to accept him and Ephialtes throws himself off a cliff in an unsuccessful suicide.

The Persian army arrives and various encounters are fought. Every battle proves a decisive victory for Leonidas and his three hundred Spartans, and the Persian army suffers horrible and constant losses. Then Ephialtes regains consciousness and, distraught at Leonidas' rebuke, seeks out the Persian king Xerxes to inform the king of a hidden mountain pass which leads around the Hot Gates. Within hours, elements of the vast Persian army cross around the Hot Gates and encircle the Greeks. Aside from a handful of exceptions, all non-Spartan Greeks desert their post as everyone realizes that to remain behind means certain death.

Leonidas exults, realizing his name will be immortalized and his death will rally not only Sparta, but all of Greece, to a common cause. He dispatches the Spartan Dilios, a wounded storyteller, to escape from the Hot Gates and carry the news to Sparta. In a final battle Leonidas and his three hundred perform their honorable duty with distinction before being killed to a man. As Leonidas predicted, their signal action galvanizes Greek resistance. The graphic novel concludes with Dilios leading a company of Spartans into battle at Plataea. Prior to charging into combat, he encourages his men by recounting the story of Leonidas at the Hot Gates and then recalling the recent Athenian decisive naval victory at Salamis.