We meet Brooklyn teen Miles Morales (voice of Shameik Moore) as he is getting ready to start attending a private school. He lives with his father Jefferson (voice of Brian Tyree Henry) and mother Rio (voice of Luna Lauren Velez). Miles goes around his neighborhood greeting his friends and tagging street signs with stickers he made. Jefferson, a cop, catches Miles and escorts him to school in his cruiser. As they ride to the school, they see a news report on Spider-Man, whom Jeff is not a fan of. Once they arrive, Jeff tells Miles he loves him, but Miles fails to say it back. Jeff then uses his radio to force Miles in front of all the other students to say "I love you" back, embarrassing him.
Miles gets into his schoolwork as he tries to adjust to this new environment. During one of his classes, he meets Gwen Stacy (voice of Hailee Steinfeld), who doesn't immediately tell him who she is. Later, Miles sneaks out of his dorm to visit his uncle Aaron (voice of Mahershala Ali), who is Jefferson's brother and a sort of black sheep due to his criminal activities. Miles tells him about Gwen, and Aaron tells him to do a shoulder touch to try and charm her. The two then go to the tunnels near the subway so that they can do some graffiti, since Aaron is fond of Miles's artwork. As Miles spray-paints the walls, a radioactive glitching spider crawls up his leg and bites his hand after Miles takes a picture of his work, but he only lightly taps the spider off his hand before leaving with Aaron.
The next day, Miles finds himself feeling differently. He hears a voice in his head (which is accompanied by comics-style text boxes), his clothes barely fit, and he is sweating profusely. He runs into Gwen, who tries to make up a fake name when they properly introduce themselves. Miles tries to do the shoulder touch, but he only gets his hand stuck on Gwen's shoulder, and then her hair, leading them to have to go to the nurse for her to cut her hair off. When Miles tries going back to his room, he is found by a security guard who calls him out for leaving his dorm. Miles runs and hides in the man's office, getting everything stuck to him as he keeps freaking out. He climbs out the window and finds himself walking on the walls before running into the streets, amazed by his newfound powers and abilities.
Miles later goes to the tunnel where the spider bit him. As he examines it, it starts glitching again. Not long after, Miles hides as his spider sense detects danger. Spider-Man enters as he's being pursued by the Green Goblin (voice of Jorma Taccone). The fight takes them beneath Fisk Industries where Wilson Fisk/Kingpin (voice of Liev Schreiber) is attempting to start up a Super Collider. In the middle of Spidey fighting Goblin, he takes the time to get Miles out of harm's way. Spidey realizes Miles is just like him, just as the machine is turned on. Outside in the city, certain structures start to get weirdly morphed into other shapes. A blast occurs in the tunnel. Spidey is badly injured, and Miles tries to help him, but Spidey tells him to hide before giving him a drive to shut down the Collider. Kingpin, Goblin, and another villain, The Prowler, gather around Spidey and remove his mask. He begs Kingpin not to restart the Collider, even telling him to think about his family, but this angers Kingpin and he kills Spider-Man by slamming his fists down onto him, which Miles watches in horror. He runs back home where his parents find him, and he runs to hug Jefferson.
The news breaks out that Spider-Man is dead, and that he was Peter Parker. New Yorkers everywhere are devastated. Miles buys a costume (the store owner is the voice of Stan Lee, no less) before attending a memorial service for Peter, with many other fans attending dressed as the wall-crawler. Mary Jane (voice of Zoe Kravitz) delivers a eulogy for her husband while Aunt May (voice of Lily Tomlin) stands solemnly in the back.
Miles is inspired by MJ's words to take up the mantle as Spider-Man. He wants to test out his powers, but he isn't quite sure of how to get them to work. He later pays a visit to Peter's grave, just as he is spotted by another man...Peter B. Parker (Jake Johnson). In his own words, this Peter tells us that in his universe, he's been Spider-Man for years and is now an adult, but he's lost Aunt May and he was married to Mary Jane but is now divorced due to him not being able to work out his issues as Spidey, as well as him not wanting kids when she did. He became depressed and gained weight, and then the Collider turned on, which pulled him into this dimension. Now after finding Miles, he gets knocked unconscious, leading Miles to try and swing away with him as the police go after them, only to end up crashing on the streets as people walk past them.
