Scientist Richard Parker (Campbell Scott) records a video message detailing his mysterious disappearance, but prior to this, he destroys much of his research for Oscorp on spiders and genetic mutations. Unfortunately, while flying on a private jet with his wife Mary (Embeth Davidtz), their journey takes a perilous turn when an assassin, sent to eliminate Richard, hijacks the plane, killing the pilot and shooting Mary during a fierce struggle for control of Richard’s laptop. Richard heroically ejects the assailant but is left to face a tragic fate as the plane crashes, prompting him to upload all his critical research to a satellite server named Roosevelt.
Fast forward to the present day, Richard and Mary’s son Peter Parker (Andrew Garfield), now a crime-fighting Spider-Man, is on a mission to protect his city. He swiftly captures Aleksei Sytsevich (Paul Giamatti), who attempts to steal plutonium vials from an OsCorp truck. In a daring rescue, Peter saves a terrified Max Dillon (Jamie Foxx), who found himself in the truck's path while it rampaged through the streets of New York, and ensures the precious cargo remains intact.
During a phone conversation with Gwen Stacy (Emma Stone), Peter catches a glimpse of her late father, George Stacy (Denis Leary), who warns him to keep Gwen out of the dangers that come with being Spider-Man. His first attempt to keep his promise results in him arriving late to his graduation ceremony, where he surprises Gwen with a passionate kiss on stage.
After graduation, Peter and Gwen meet at a restaurant, where Peter insists on honoring his vow to her father to stay away from her, leading to an emotional breakup as Gwen seeks new opportunities at Oxford University. In addition, Peter juggle life as Spider-Man while grappling with his increasingly burdensome secret from his aunt, May (Sally Field).
Meanwhile, Max, an engineer at Oscorp, finds fascination in Spider-Man due to his past rescue and inadvertently becomes a living electric generator after an accident involving genetically modified eels. This incident draws attention to the ethically dubious dealings at Oscorp, with Max receiving scant concern from senior VP Menken ([Colm Feore]), who plots to eliminate Harry Osborn's control over his family’s company.
Harry Osborn ([Dane DeHaan]), returning to Manhattan to take care of his dying father, is soon thrust into a leadership role at Oscorp after Norman ([Chris Cooper]), his father, passes away. As his illness progresses, Harry’s desperation for a cure leads him to seek help from Spider-Man, but the resulting fallout turns him into a villain.
The tension escalates as Max, now empowered and emotionally unstable, causes chaos in Times Square, forcing Spider-Man to intervene. Harry's downfall from CEO and eventual transformation into a monstrous Green Goblin strikes a significant blow to Peter, culminating in Gwen's tragic death during a confrontation at a clock tower, a devastating blow that shatters Peter's resolve and prompts him to abandon his superhero duties.
As time progresses, the villainous Rhino resurfaces as a new threat, and haunted by grief, Peter finds renewed inspiration from Gwen's hopeful graduation speech, pushing him back into the fray as Spider-Man, ready to defend his city once more against escalating dangers.
