Set in a different series of events, this story does not follow the previous Appleseed movie canon (despite being directed by the same director of the previous movies); it is an alternate story to their beginnings. Briareos is already a cyborg and did not become separated with Deunan to be later reunited in Olympus to join ESWAT. Appleseed Alpha depicts the early days of Deunan Knute and Briareos in the 22nd century. Similar to the original story, there was a world war and nobody won; governments had destabilized and cities had fallen into ruin. The city of Olympus is the unseen city of legend and dreams. Deunan Knute and Briareos are former soldiers and now work as mercenaries, dreaming of a better life. Their story begins within the ruins of New York City.
Given a job by a local despot who calls himself Two-Horns, Deunan Knute and Briareos are making a delivery run for Two-Horns. Minding a case of vaccine(s), the duo travel through the old MTA subway system to avoid trouble, but were ambushed by cyborgs; the two defeated their enemies and made it to ruins of Times Square. Worried that Two-Horns might get the wrong idea that they ran for it, Briareos and Deunan returns to see Two-Horns and explained they had complications and the mission was a failure. Despite the failed mission, as far as the two are concerned, all their debts to Two-Horns are repaid and they want out of their operations. However, Two-Horns values them and refuses to let them off that easily. It was Two-Horns that secretly sabotaged his own assignment, hoping to use it as a means to extend the duo’s service to him.
Despite Two-Horns’ displeasure at the situation, things change when Briareos experiences internal system problems. Deunan reluctantly agrees to help Two-Horns for one last job to help get Briareos patched up. Assigned to the outskirts of the city to clear up some old war drones, the duo were attempting to clear the drones when they encountered a transport driving straight into the target zone with the drones. The duo jump in and saved a cyborg soldier and bioroid girl, Iris (voiced by Brina Palencia) and Olson.
Grateful for being saved, Olson (a cyborg soldier), helps troubleshoot Briareos’ malfunctioning systems and Briareos helps them get their Humvee working again. Both groups distrusted each other, but because there is no cause for alarm, the two groups traveled together while figuring out their next move(s). Shortly afterwards, a powerful cyborg named Talos, leader of the Triton faction (a group of highly skilled and advanced cyborgs), arrived in pursuit of Olson and Iris. Talos has grand ambitions to remake the ruined world in his image; he needs Iris to help him complete his quest. He arrives on the scene to investigate his targets of interests, but only to find the remains of combat. When the four realize Triton is after them, they decided it best to stick together until their situation is resolved. Olson knows of Talos’ reasons for hunting them, but couldn’t tell Deunan nor Briareos due to his classified mission.
Talos was an agent of Olympus until he developed a messiah complex and defected; he planned to rebuild the world in his image. He previously had Two-Horns work for him, but after discovering Deunan and Briareos had Olson and Iris, he suspects Two-Horns of betrayal. Talos confronts Two-Horns about his men, but Two-Horns claims innocence. Although Talos believes him, he feels their partnership is at an end and had his second, Nyx, finish off the gang by destroying their building. Although Two-Horns’ base is destroyed, he and his gang survived and decided to take their fury upon Deunan and Briareos. Two-Horns and his men discovered and attacked the duo with a battle tank, but Olson and Iris help stopped them; it is then that Talos arrives and arrested Iris and Olson under his custody and flies off.
Talos was looking for Iris and Olson due to their classified mission from Olympus: to destroy an abandoned super war machine. He forcefully downloads the data from Olson and throws him out of the plane, killing him, but not before Olson leaves a clue for Deunan and Briareos to find. Briareos locks onto Olson’s radio frequency and finds Olson’s body; he had downloaded a chip that explains their mission and the duo now searches for Iris. Talos eventually finds the abandoned secret underground hangar that holds a giant arachnid-themed mobile battle fortress, the very source of power he wanted to help remake the world. However, Deunan and Briareos intervened.
Two-Horns had survived his last fight and decided his real target is Talos; he sided with Deunan and Briareos. Two-Horns helps them take on Talos’ cyborgs by providing weapons and an Landmate for Deunan to fight in. Talos uses Iris to activate the sleeping giant, but the machine detected a hull breach (Deunan fought her way through the behemoth’s defense system at the cost of her Landmate) and goes on the offensive and targets the remains of New York City. Briareos tries to assist Deunan, but is confronted by Nyx; he kills her in melee combat. Deunan made it inside the mobile fortress and battles Talos. Deunan shot Talos down, but he gets up again and grabs Deunan, with the help of Iris, Talos is crushed to death by a blast door. Deunan and Briareos, manage to destroy the giant, but at the price of Iris sacrificing her life. After the long battle, Two-Horns accepts the duo’s departure and offers Deunan the repaired Landmate as a parting gift. The two ride off to find Olympus.
In the post credits, Olympus Officer Hitomi (Iris’ original clone template) is speaking with another officer. They confirmed the destruction of the prototype super-weapon and the officer asks Hitomi about retrieving Deunan and Briareos to Olympus. However, Hitomi believes the duo are like Appleseeds, wherever they travel, they will bring hope and others will follow in their wake; Hitomi wants them to do more good in the bad lands before taking them in.