The Asgardian Loki encounters Chthon, the leader of an extraterrestrial race known as the Chitauri. In exchange for retrieving the Tesseract, a powerful energy source of unknown potential, Chthon promises Loki an army with which he can subjugate Earth. Nick Fury, director of the espionage agency S.H.I.E.L.D., and his lieutenant Agent Maria Hill arrive at a remote research facility during an evacuation, where physicist Dr. Erik Selvig is leading a research team experimenting on the Tesseract. Agent Phil Coulson explains that the object has begun radiating an unusual form of energy. The Tesseract suddenly activates and opens a wormhole, allowing Loki to reach Earth. Loki takes the Tesseract and uses his scepter to transform Selvig and several agents, including Clint Barton, into powerful Heartless known as the Masters of Evil to aid him in his getaway.
Back in the Hundred Acre Woods, yet another first day of Autumn has arrived; to Winnie the Pooh it’s another fun-filled season to be with his very best friend, Christopher Robin. However, he is nowhere to be found, which causes Pooh to worry what happened to him. After finding a sticky note on top of a hunny pot, Owl scares Pooh, Piglet, Tigger, Rabbit, Eeyore, Derrick, Joshua, Bob, Helen, Violet, Dash Parr, the Road Rovers, Olaf, Elliot, Timon, Pumbaa, Otto, Mr. Rat, Mr. Mole, Benjamin, Bongo, Lullubelle, Sora, Mickey and Minnie Mouse, Donald Duck, Goofy, Louis, the Gummi Bears, Belle, Bambi, Faline, Tod, Copper, Kenai, Genie, Turbo, Dumbo, Wreck-It Ralph, Vanellope von Schweetz, Ash Ketchum, Simba, and their respective team members when he reveals that, according to the note, Christopher Robin has been kidnapped by the Evil Schemers, with the hunny pot being a sort of Faux Affably Evil parting gift of friendship to twist the knife in further. Pooh, Ash, Simba and the others go to Master Yen Sid. He talks that the Evil Schemers have returned. Pooh, Ash, Simba and the others decide to head out to find and save their friend.
In response to the attack, Fury reactivates the "Avengers Initiative". Agent Natasha Romanoff is sent to Calcutta to recruit Dr. Bruce Banner to trace the Tesseract through its gamma radiation emissions. Coulson visits Tony Stark to have him review Selvig's research, and Fury approaches Steve Rogers with an assignment to retrieve the Tesseract. In Stuttgart, the Heartless of Barton, now calling himself Trick Shot of HYDRA, steals iridium needed to stabilize the Tesseract's power while Loki causes a distraction, leading to a confrontation with Rogers, Stark, and Romanoff that ends with Loki's surrender. Christopher Robin is rescued by Blitz. While Loki is being escorted to S.H.I.E.L.D., Thor, his adoptive brother, arrives and frees him, hoping to convince him to abandon his plan and return to Asgard. After a confrontation with Stark and Rogers, Thor agrees to take Loki to S.H.I.E.L.D.'s flying aircraft carrier, the Helicarrier. There Loki is imprisoned while scientists Banner and Stark attempt to locate the Tesseract.
The Avengers become divided, both over how to approach Loki and the revelation that S.H.I.E.L.D. plans to harness the Tesseract to develop weapons as a deterrent against hostile extraterrestrials. As the group argues, Trick Shot and Loki's other possessed agents, transformed into doppelgangers of Living Laser, Grey Gargoyle, Radioactive Man, Whirlwind, Dreadknight, and Hypnotia, attack the Helicarrier, disabling its engines in flight and causing Banner to transform into the Hulk. Stark and Rogers try to restart the damaged engine, and Thor attempts to stop the Hulk's rampage. Romanoff fights Trick Shot, only for himself to fully become possessed by an avatar of Loki and unleashing the powers of darkness upon her in an attempt to break her soul, but she is saved from Loki-Trick Shot by Sora, who uses his Keyblade to stab the possessed agent in the heart, causing him to blow up in dark energy and revert back into the real Barton, now free of Loki's avatar. Speaking of which, Loki escapes after killing Coulson and ejecting Thor from the airship, while the Hulk falls to the ground after attacking a S.H.I.E.L.D. fighter jet. Fury uses Coulson's death to motivate the Avengers into working as a team. Stark and Rogers realize that for Loki, simply defeating them will not be enough; he needs to overpower them publicly to validate himself as ruler of Earth. Loki uses the Tesseract, in conjunction with a device Selvig built, to open a wormhole above Stark Tower to the Chitauri fleet in space, launching his invasion. The Chitauri soon bow themselves in allegiance to the Masters of Evil, who band together to wreak mayhem in New York for the cause of world domination.
The Avengers rally in defense of New York City, the wormhole's location, but quickly realize they will be overwhelmed as wave after wave of Chitauri descend upon Earth. Banner arrives and transforms into the Hulk, and together he, Rogers, Stark, Thor, Barton, and Romanoff battle the Masters - Chitauri alliance while evacuating civilians. The Hulk finds Loki and beats him into submission. Romanoff makes her way to the wormhole generator, where Selvig, having been reverted back from his previous Heartless form of the Leader, reveals that Loki's scepter can be used to shut down the generator. Meanwhile, Fury's superiors attempt to end the invasion by launching a nuclear missile at Manhattan. Stark intercepts the missile and takes it through the wormhole toward the Masters of Evil. The missile detonates, destroying the Chitauri mothership and disabling their forces on Earth, as well as causing the Masters to convulse and die in dark energy explosions, freeing the slain SHIELD agents from the scepter's power. Stark's suit runs out of power, and he falls back through the wormhole just as Romanoff closes it. Stark goes into freefall, but the Hulk saves him from crashing to the ground. In the aftermath, Thor returns Loki and the Tesseract to Asgard. Fury expresses confidence that the Avengers will return if and when they are needed.
In a mid-credits scene, Chthon confers with his master, revealed to be Thanos the Mad Titan. about the failed attack on Earth and humanity's strength; in a post-credits scene, the Avengers eat in silence at a shawarma restaurant.
- Wreck-It Ralph, Dumbo, Simba, Donald Duck, Goofy, Sora, Bob Parr, Helen Parr, Violet Parr, Dashiell Robert Parr, Ash Ketchum, Misty, Brock, Togepi, Pikachu and The Evil Schemers will guest star in this film.
- This film takes place after Ash Ketchum Meets Dumbo (which explains Ash already know Dumbo) and Winnie the Pooh Meets The Incredibles (which explains Winnie the Pooh, Ash and friends met the Parr family).
- The villians will work for Loki.
- In the end of the film Pooh and Pals have been made official Avengers.