On the watercolor-reminiscent Earth-65, Gwen Stacy (voice of Hailee Steinfeld) is struggling to live up to the expectations of her father, police captain George Stacy (voice of Shea Whigham), who does not know that she is Spider-Woman. He is hunting her after mistakenly believing that she caused the death of her best friend, Peter Parker. One night, Gwen heads over to the Guggenheim Museum and encounters a version of the Vulture from an Italian Renaissance-themed alternate universe. Miguel O'Hara (voice of Oscar Isaac) and Jessica Drew (voice of Issa Rae) arrive from yet another alterate universe using portal-generating watches and help Gwen neutralize Il Avvoltoio, who is then sent back to his home dimension. George corners Gwen, who then reveals her identity to him; distraught, he attempts to arrest her. Miguel reluctantly grants Gwen membership into the Spider-Society, allowing her to escape with him and Jess.
In Brooklyn on Earth-1610, sixteen months after the Alchemax collider's destruction, Miles Morales (voice of Shameik Moore) is adapting to being Spider-Man while internally handling his feelings for Gwen and struggling to live up to his parents' expectations. While heading to a parent-teacher evaluation, he encounters the Spot (voice of Jason Schwartzman), a scientist infused with portals after the Alchemax collider explosion. The Spot blames Miles for causing his condition and reveals that, while testing the collider, he had transported a spider from another dimension, Earth-42, which bit Miles and turned him into Spider-Man. The Spot then accidentally transports himself into a void, where he learns to travel to other universes that contain the Alchemax collider so he can use them to empower himself.
Gwen travels to Earth-1610 and she and Miles reconnect, though she is secretly tracking the Spot across dimensions. Miles invisibly watches Gwen trace him, after which Jessica contacts Gwen and instructs her to leave Miles behind. Gwen opens a portal, which Miles follows through to Mumbattan on Earth-50101. They team up with local Spider-Man Pavitr Prabhakar (voice of Karan Soni), and later Hobie Brown, Spider-Punk (voice of Daniel Kaluuya) in an attempt to stop the Spot from absorbing the powers of that reality's Alchemax collider. Miles is struck with a vision of his father Jefferson (voice of Brian Tyree Henry), who is soon to be promoted to captain, dying at the hands of the Spot before the Spot escapes.
In the ensuing destruction of the collider, Miles saves a police captain, the father of Prabhakar's girlfriend. However, Mumbattan then begins falling apart from the disruption of a "canon event". Members of the Spider-Society arrive to assess the damage of the dimensional anomaly, while Miles, Gwen, Prabhakar, and Brown are sent to the Spider-Society headquarters on Earth-928, where hundreds of Spider-Man variants reside.
They meet up with Miguel, and are joined by Peter B. Parker (voice of Jake Johnson) and his daughter Mayday. Miguel explains to Miles that, though the details of each Spider-Man's story changes with the details of their reality, all of them involve The Stations of the Canon, such as the deaths of a police captain close to Spider-Man and a parental figure to Spider-Man. These events define not only the character but that Alternate Timeline; straying from them may cause the continuity to collapse, and too many of those will bring The Multiverse crashing down as well. Miguel reveals he witnessed the collapse of another universe firsthand while attempting to replace a deceased version of himself from that universe, and has since attempted to keep all of creation from unraveling. To do this, he assembled the Spider Society and has been patrolling ever since, apprehending people who have breached dimensions (including a version of Aaron Davis who is being played by Donald Glover in live action) and sending them home.
Miles realizes that the death of Jefferson Morales — a parental figure to Spider-Man; a police captain close to Spider-Man — is a canon event.
Miguel imprisons Miles to prevent him from saving Jefferson, but Hobie helps Miles break loose before quitting the Society. Miguel orders all of the Spider-People to apprehend Miles, resulting in a long chase through the complex. Miguel eventually corners Miles and reveals that Miles himself is the Butterfly of Doom that is threatening to collapse the Multiverse: the spider that bit him came from Earth-42, resulting in Earth-42 having no Spider-Man and the death of Earth-1610's Peter Parker (the one played by Chris Pine), not to mention the creation of the Spot. Gwen and Peter B. reveal they kept Miles Locked Out of the Loop regarding his father's death and Miles' status as the original anomaly. Miguel insists that Miles stand by and let his father die in service of The Needs of the Many, but Miles refuses:
- Everyone keeps telling me how my story is supposed to go! Nah: I'mma do my own thing.
Miles escapes Miguel and uses the Spider Society's "Return To Home Dimension" machine to go home and save his father. Seeing Gwen as a liability, a furious Miguel kicks her out of the Spider-Society and sends her back to her universe. Gwen is powerless to stop him, but uses her last words as an Armor-Piercing Response:
- We are supposed to be the good guys.
Gwen reconciles with her father, who admits he has decided to resign as police captain. Realizing that his resignation is evidence that a timeline can go Off the Rails without going Off the Rails, Gwen decides to aid Miles, leaving Earth-65 with a bootleg portal watch Hobie left with her father for her.
As Miles arrives back in his apartment, he reveals to his mother that he is Spider-Man. She has no idea who that is. Miles realizes the Spider Society machine sent him to Earth-42, where his uncle Aaron (voice of Mahershala Ali) is alive, Jefferson Morales is dead, and because Spider-Man does not exist, New York City is riddled with unchecked crime. Aaron interrogates Miles and is soon joined by Earth-42's The Prowler... none other than Miles Morales himself (voice of Jharrel Jerome). The Prowler refuses to release Miles to go save his father.
As Miguel, Jessica, and Ben Reilly search for Miles, Gwen travels to Earth-1610 and speaks with Miles' parents, promising to find him and bring him home. As the Spot begins his multiversal attack, Miles begins prepping his venom-strike powers to escape, while Gwen assembles a team — Peter B., Mayday, Pavitr, Hobie, Margo Kess, Spider-Man Noir, Peni Parker, and Spider-Ham — to go help her friend.
