Deunan Knute is a young highly skilled female soldier and is one of the last survivors of the Global War. During a battle, she is rescued by Hitomi, a Second Generation Bioroid. Deunan attempts to escape from a hospital and takes Hitomi hostage, but is stopped by Briareos Hecatonchires, her former lover, who is now a cyborg. She learns that the war had ended and she is in a Utopian city called Olympus. Its population is half-human and half-clone, a genetically-engineered species called Bioroids. Olympus is governed by three factions: Prime Minister Athena Areios; General Edward Uranus III, head of the Olympus Army; and a Council of Elders. Everything in the city is observed by an artificial intelligence named Gaia from a building called Tartaros. While there, Deunan joins the ESWAT organization.The Bioroids were created from the DNA of Deunan's father, Carl, making the Second Generation Bioroids her brothers and sisters. However, they have a much shorter lifespan than humans due to suppressed reproductive capabilities. The Bioroids life extension facilities are destroyed by a secret faction of the Regular Army in a terrorist attack against the Bioroids. However, the Appleseed data, which contains information on restoring the Bioroids reproduction capabilities, still exists.Olympus is plagued by conflicting factions. Along with a strike force, Deunan and Briareos head to the building where the Bioroids were originally created. She activates a holographic recording showing the location of the Appleseed data. Dr. Gilliam Knute, who created the Bioroids, entrusted Appleseed to Deunan, but was shot by a soldier. Colonel Hades, the anti-Bioroid terrorist, reveals to Deunan that Briareos had intentionally allowed his Landmate, a large exoskeleton-like battlesuits, to escape. They run to the roof where their team is killed and they are cornered by the Regular Army. Uranus tries to convince Deunan that Bioroids seek to control humanity, and he wants to destroy D-Tank full of virus material in Tartarus. Briareos tells Uranus that the Elders were the ones who planned this; the D-tanks virus was originally believed to keep Bioroids in line, but in actuality is capable of sterilizing humanity. Hades convinces Uranus that negotiating is pointless, and blames Carl for all that has happened before Deunan kills him. They manage to escape, and later wash up on a beach. Briareos, badly damaged in the firefight, lays dying in the sand. Grief-stricken, Deunan refuses to leave his side. Briareos confeses everything to Deunan, hoping to coax her into continuing on. Mechanic Yoshitsune Miyamoto arrives in his Landmate and begins repairing Briareos. Deunan takes the Landmate back to Olympus, where the Appleseed data is used to fully restore Bioroid reproductive functions.Despite the relief, Deunan confronts the Council of Elders and force them to admit that they killed her mother. They reveal they plan to use the D-tank virus to sterilize all humans, allowing their race to die out slowly, leaving the Bioroids the new rulers of Earth. They needed the Appleseed data to keep the Bioroids alive, but Gilliam hid the data so they could not move forward with their plan. Athena, stepping in to stop them and announcing that Uranus has surrendered, tells Deunan that the Elders had been acting on their own and had shut Gaia down once they realized humanity had softened their stance against Bioroids. The Elders state that they will soon die since Gaia kept them alive, but that they were ready to sacrifice themselves. They then activate the city's mobile fortress defenses, which begin marching towards Tartaros. Athena states that D-tank's security system is nearly impenetrable, but a shot from the fortresses' main cannons might puncture the tank, releasing the virus. ESWAT begins mobilizing, but suffer heavy casualties due to the fortresses' heavy weaponry.Briareos arrives and asks Deunan to join the battle. Despite the Elders objections, Deunan goes with Briareos to the towers. Deunan attempts to enter the password to shut the defenses down, but a malfunction makes it difficult to complete. The final password letter appears by itself, and Deunan secures the D-Tank.The film ends with Deunan stating that the sins of humanity will probably get worse, but that there is always the chance that future generations will learn from these mistakes. She vows to keep fighting for the children, who are "the true new race of man."