The story begins with a mysterious rift opening in the skies above New York City, attracting the attention of Providence. Meanwhile, Rex is sparring against Agent Six, while musing about his need for a "Theme song" (and briefly performing a take on the original Ben 10 theme song) before getting the alert about the rift. Providence, at the same time, is unable to do anything before Rex arrives with Six and Bobo. Rex attempts to touch the rift to see if it has something to do with nanites when Holiday then gets a scan that shows something is coming through the rift, and Rex finds himself face to face with Humungousaur, who knocks Rex back into the park beneath them. During their skirmish, a strange metallic creature also appears and heads into the city, with Bobo and Six on its trail while Rex continues to battle Humungousaur. Just when Rex thinks he has the upper hand on the monster, it changes in a flash of light to become Diamondhead. Rex attempts to cure Diamondhead but to no avail, which surprises Rex.
Meanwhile, Six engages the creature and manages to cut a piece of it off, and when Holiday's scans give insufficient data, is given to Rex's older brother, Caesar. The hero's battle then winds up on the Brooklyn Bridge after Rex lands a well placed punch. Diamondhead gets and changes into Lodestar, attempting to counter his opponent's metal abilities. However Rex quickly adapts and blasts Lodestar with his Slam Cannon into a Billboard. It breaks free of its binding, which forces the two to stop fighting, with Lodestar becoming Rath to save news reporter, Diane Farah while Rex saves the other civilians. Rex then literally gets the drop on Rath and once again attempts to cure him of his nanites, only for it not to work again. After attempting to explain himself, Rath decides to turn back to his true form, Ben Tennyson. Ben is shocked to learn that no one knows about him. Just then, Six appears with the mechanical creature from before, which self-destructs and renders him comatose and Rex mortified and angered.
In Providence HG's infirmary, Holiday explains that the creature was Nanite in origin, but somehow completely different from any EVO they have ever encountered. White Knight points out that part of it got away and that they were now searching for it. Ben says he knows where its going and then suggests that they are dealing with a parallel universe issue. Holiday doubts this assumption, stating that parallel universe is just a theory to which Ben replies that he has travelled to many of them. Rex threatens Ben and explains that there is no way Ben can escape, but Ben manages to use Big Chill to do so, and flies off with Rex in hot pursuit. Ben visits the area where Bellwood should be, and finding that himself, Gwen, Max, and Kevin do not exist in this world, nor does Mr. Smoothy (instead it is "Bob's Biscuit Barn"), Ben finally comes to grasp that he must be in an alternate dimension, and Rex and he finally come to a truce. Caesar then appears and seemingly prepares to attack the two with a ray gun, which was actually intended for the energy ball, which is revealed to be the Alpha Nanite, the first nanite, and a creation of Caesar's. The Alpha Nanite then begs Caesar to help him stabilize a new body to which Caesar refuses. Ben, as Diamondhead, knocks Alpha away from Caesar and manages to force Alpha to leave.
After escaping the creature, Caesar explains that he created Alpha as a means of controlling the nanites during the Nanite project, but it's the nanite form which is unsustainable and every body it builds burns out in short amount of time. When Alpha attempted to take organic bodies, this forced Caesar to banish it to another empty dimension.
Ben surmises it must have been the Null Void where it wound up, and that Alpha must have possessed alien life to sustain itself. Caesar explains that the damaged ray gun was to stop Alpha, who then attacks and destroys his research pod. Caesar jetisons the escape pod, leaving behind the two heroes. Ben saves Rex by becoming Cannonbolt, to which the latter complains of the smell. Alpha then attacks Providence in order to absorb the E.V.Os inside for their nanites. When Ben and Rex arrive, they fight the Alpha where it attempts to absorb Rex's Omega Nanite but fails. Ben and Rex eventually manage to drive Alpha off, and then slowly bond after sharing their origins during a small game of hoops.
White Knight then reveals that Alpha has gone to the Bug Jar, which is the largest concentration of active nanites on the planet after the Nanite Event. White Knight and the heroes arrive, only to find they are too late to stop Alpha from absorbing the nanites of all the EVOs that were in the Bug Jar to become a colossal monster that begins to attack the three of them. Alpha is unable to absorb Rex's Omega Nanite so he decides to hack Ben's Ultimatrix which gives him access to his own Nanite corrupted version of it and 3 of Ben's strongest aliens, Heatblast, Four Arms, and Humungousaur. During the battle, Ben gains the form of Shocksquatch, which only lasts a few seconds but enough to stop Alpha momentarily. Using an odd form of teamwork, Rex is able to disable Alpha's imitation Ultimatrix powers, but is ensnared by his enemy and robbed of his Omega Nanite, leading to Nanite Alpha to transform into a colossal crab-centaur-like being calling itself Alpha-Omega; the beginning and the end.
As the now nearly unstoppable Alpha-Omega rampages through the Bug Jar; synthesizing the ruined structures in order to create new nanites for itself, Rex has doubts about himself now that his most powerful builds are gone. Ben however comes up with a plan, and transforms into Upgradeand merges with Rex, After a couple seconds, The duo then take on the Alpha-Omega, to which they hold their own for a short while. At the last minute, Rex and Upgrade do a final bout of teamwork, manage to nail Alpha-Omega directly in his Omega Nanite, which forces the monstrosity into a large cloud-like entity. Rex then use his ability to begin to densen the nanite cloud into a ball. While this happened, Caesar appears and extracts the Omega Nanite. Attempting to dispose of Alpha, Caesar then opens a rift for Ben, who changes into Murk Upchuck and swallows the compacted Alpha as he departs to the Null Void and back to his own Earth, but not before weirding out Rex one last time.
In the epilogue, Rex is overjoyed that Six has recovered, and wonders if he'll ever see Ben again. Six suggests that they possibly will, if fate had ordained for them to meet once already. Caesar appears and reinjects the Omega Nanite into Rex, who fears that it may actually be the Alpha Nanite, although Caesar assures him that there is a 99.998% chance that it is Omega. Finally, in the depths of the Null Void, Alpha begins to stir within its prison of matter, causing it to glow and crack apart.
- Ash Ketchum, Pikachu, Serena, Misty, Togepi, Brock, Littlefoot, Cera, Ducky, Petrie, Spike, Scooby-Doo, Shaggy Rogers, Fred Jones, Daphne Blake, Velma Dinkley, The DigiDestined and their Digimon, Kazuto Izuka, Narue Nanase, Masaki Maruo, Hajime Yagi, Kanaka Nanase, Mr. Peabody, Sherman, Penny Peterson, Serena and the Sailor Scouts, Lincoln and his sisters, The Human Mane 5, The Bowser Family, Dr. Facilier, The Grand Duke of Owls, Attila and Hun, The Dazzlings, Megan, Kurumi Tokisaki, The Crime Empire (Joker, Harley Quinn, Two-Face, Penguin, Negaduck, Megavolt, Quackerjack, Bushroot, and The Liquidator), Kyoko Kudo, Yuki Kashiwazaki, and Aya Kanazawa and Team Rocket (Jessie, James, and Meowth) guest stars in this film.
- The Bowser Family, Dr. Facilier, The Grand Duke of Owls, Attila and Hun, The Dazzlings, Megan, Kurumi Tokisaki, The Crime Empire, Kyoko Kudo, Yuki Kashiwazaki, and Aya Kanazawa and Team Rocket will work for Alpha in this film.
- Chris Brown - Wall to Wall will be used in the End Credits.
- Yuri Lowenthal as Ben Tennyson, Upgrade
- Daryl Sabara as Rex Salazar
- Dee Bradley Baker as Humungousaur, Diamondhead, Lodestar, Big Chill, Upchuck
- John DiMaggio as Rath, Bobo Haha, Black Pawn
- David Kaye as Shocksquatch
- Grey DeLisle as Rebecca Holiday, Diane Farrah
- Wally Kurth as Agent Six
- Jennifer Hale as Black Knight
- J.K. Simmons as White Knight
- Freddy Rodriguez as Caesar Salazar
- Michael Emerson as Alpha