Deunan Knute, a talented and resilient female soldier, finds herself among the last remnants of humanity after a brutal Global War. In a twist of fate during a skirmish, she is saved by Hitomi, a Second Generation Bioroid. Trying to flee from a hospital, Deunan takes Hitomi captive, only to be confronted by her former lover, Briareos Hecatonchires, who has since transformed into a cyborg.
As she comes to grips with her new surroundings, Deunan learns that the war is over, and she is now in Olympus, a supposedly Utopian city inhabited by both humans and Bioroids—genetically-engineered beings created from the DNA of her father, Carl. Olympus is governed by three powerful entities: Prime Minister Athena Areios, General Edward Uranus III of the Olympus Army, and a Council of Elders. Overseeing this intricate society is an artificial intelligence named Gaia, residing in a structure known as Tartaros.
In this new reality, Deunan joins the elite ESWAT organization, uncovering troubling truths about the Bioroids’ creation and their dangerously curtailed lifespans due to their engineered inability to reproduce. A tragic terrorist attack from a rogue faction of the Regular Army leads to the destruction of the life-extension facilities meant for the Bioroids. Yet, a glimmer of hope lies in the Appleseed data, which possesses crucial information to restore their reproductive capabilities.
Tension escalates in Olympus as Deunan navigates the treachery among different factions. Alongside Briareos, she embarks on a perilous mission to the facility where the Bioroids were first developed, hoping to locate the elusive Appleseed data. A holographic projection reveals the data's whereabouts, but complications arise when Dr. Gilliam Knute, the Bioroids' creator and Deunan's father, gets fatally shot by a soldier. Meanwhile, Colonel Hades, a fierce opponent of the Bioroids, exposes a sinister plot where Briareos deliberately allowed a colossal exoskeleton, known as a Landmate, to escape.
In a heart-pounding encounter, Deunan and her team face overwhelming odds against the Regular Army. General Uranus attempts to persuade Deunan that Bioroids aim to dominate humanity, revealing plans to obliterate a D-Tank filled with toxic virus material located in Tartaros. Briareos counters that the Elders orchestrated this scheme, as the D-Tank's virus was initially meant to subdue the Bioroids but has lethal implications for humanity as well. When Hades incites Uranus to abandon dialogue, he points the finger at Carl for the chaos, only to face Deunan's wrath.
After a narrow escape, they find themselves washed ashore, where a gravely injured Briareos confesses his truth to Deunan, urging her to persevere. The skilled mechanic Yoshitsune Miyamoto arrives in his Landmate, initiating repairs on Briareos, and Deunan eventually returns to Olympus, utilizing the Appleseed data to restore Bioroid reproductive capabilities.
Despite these victories, Deunan confronts the Council of Elders, demanding justice for her mother's death. They reveal their horrific plan to gradually exterminate all humans, leaving the Bioroids to rule the Earth. The Elders had sought the Appleseed data to prolong their own kind's survival, but Gilliam had hidden it for good reason. With Athena intervening to clarify that Uranus has surrendered, she informs Deunan that the Elders acted autonomously and had shut down Gaia when they suspected humanity's view on Bioroids was changing.
As the Elders prepare for their own demise, they activate the city's fortress defenses against Tartaros, setting the stage for a showdown. With ESWAT suffering significant losses against the formidable armament of the fortresses, Briareos requests Deunan's assistance. As tensions peak, she strives to enter the password to disarm the defenses, but malfunctions complicate the process. Ultimately, a final letter appears, allowing her to secure the D-Tank.
The film concludes with Deunan reflecting on humanity’s continuing imperfections, acknowledging that the future might be fraught with challenges, yet holding onto the hope that the next generations will learn from past errors. She resolutely commits to fighting on behalf of the children, whom she deems "the true new race of man."
