The film is based on the life of Alexander the Great, King of Macedon, who conquered Asia Minor, Persia and part of Ancient India. Shown in the movie are some of the key moments of Alexander's youth, his invasion of the mighty Persian Empire and his death. It also outlines his early life, including his difficult relationship with his father, Philip II of Macedon (Val Kilmer), the unification of the Greek city-states under the League of Corinth and the conquest of the Persian Empire in 331 BC. It also details his plans to reform his empire and the attempts he made to reach the end of the then known world.
The storyline begins in 356 BC with Ptolemy I Soter (Anthony Hopkins), who narrates throughout the film. With a lavish budget, the film depicts Alexander's daily life and the strained relationship between his parents. Alexander grows up with his mother Olympias (Angelina Jolie) and his tutor Aristotle, where he finds interest in love, honour, music, exploration, poetry and military combat. His relationship with his father is destroyed when Philip marries Attalus's niece, Eurydice.
After Philip is assassinated, Alexander becomes king of Macedonia and the rest of Greece. Having briefly mentioned his punitive razing of Thebes and burning of Persepolis, Ptolemy gives an overview of Alexander's west-Persian campaign, including his being declared as the son of Zeus by the Oracle of Amun at Siwa Oasis, his great battle against the Persian Emperor Darius III in the Battle of Gaugamela and his eight-year campaign across Asia.
The plot also illustrates Alexander's private relationship with his childhood friend Hephaestion (Jared Leto) and later his wife Roxana (Rosario Dawson). Before succumbing to an unknown illness or poison — it is never revealed which, yet both are suggested —, Alexander distances himself from his wife, despite her being pregnant, believing that she has killed his childhood friend Hephaestion.
The film accurately places Hephaestion in a far closer position to him than Roxanna. It is suggested in the film that Alexander died in part due to the loss of Hephaestion, which occurred less than three months earlier. Early in the film, Hephaestion compares Alexander to Achilles, to which Alexander replies that, if he is Achilles, Hephaestion must be his Patroclus (Achilles's supposed lover). When Hephaestion mentions that Patroclus died first, Alexander pledges that, if Hephaestion should die first, he will follow him into the afterlife — a promise that Ptolemy observes he apparently kept when he died shortly after his friend.
After conquering Babylon, Alexander admits that Hephaestion is the only person whom he loves. During the film, Hephaestion shows extensive jealousy when he sees Alexander with Roxana and deep sadness when he marries her, going so far as to attempt to keep her away from him after Alexander murders Cleitus the Black in India.
The film also focuses intensively on the close and warped relationship that Alexander shared with his mother Olympias, even though he never saw her after embarking on his long campaigns.