Miles Morales is a young boy of Afro-Latino heritage living in Brooklyn with his parents. He is attending a new prep school he dislikes and doesn't feel like he fits in. Miles' father is a police officer who wants Miles to follow the straight and narrow path but he is more interested in his art and hanging out with his uncle Aaron.
One night, Miles goes to an abandoned subway station to work on his graffiti art and gets bitten by a radioactive spider, giving him spider-like abilities. The next day, he stumbles upon a battle between Spider-Man and Green Goblin. Spider-Man is badly injured and gives Miles a USB drive, telling him to destroy it.
Later, Miles learns that the USB contains plans to access an inter-dimensional portal which can bring other versions of Spider-Man into his universe. Wilson Fisk, Kingpin, wants to use the device to bring back his wife and son, who died in a car accident. However, the device malfunctions and brings various other Spider-People to Miles' world.
These include Peter B. Parker, an older, out-of-shape Spider-Man from an alternate dimension; Gwen Stacy, also known as Spider-Woman; Spider-Man Noir, a version from the 1930s; Peni Parker, a Japanese girl who operates a spider-shaped robot; and Spider-Ham, a cartoon pig version of Spider-Man. They all team up with Miles to stop Kingpin and return home to their original dimensions.
Throughout the movie, Miles grapples with his newfound abilities and what it means to be Spider-Man. He also tries to balance being a superhero with his personal life, including his relationship with his father and his uncle's ties to a criminal organization. In the end, Miles embraces his role as Spider-Man and saves the day, leading the other heroes back to their own dimensions.