During his childhood, Peter Parker discovers that his father Richard's study had been ransacked. His father gathers severeal deliberately hidden documents before Peter's parents take him to stay with his Aunt May and Uncle Ben. His parents then leave, but are later killed in a plane crash. Peter is left in the care of his Aunt and Uncle indefinitly as a result.
As a teenager, Peter is a student at Midtown Science High School, where he is bullied by stereotypical tough guy Flash Thompson, and is secretly in love with Gwen Stacy, the daughter of NYPD captain George Stacy. At home, Peter discovers his father's documents, and learns he had been working with fellow Oscorp scientist Dr. Curt Connors to combine human and animal DNA to cure illnesses. Peter sneaks into Oscorp in order to meet Connors, who is pressured by his superior Dr. Rajit Ratha to complete his serum, and cure the terminally ill head of Oscorp Norman Osborn. Connors wants the cure himself to restore his missing arm. Meanwhile, Peter breaks into a lab where a "biocable"-a high strength strand of webbing-is created by genetically modified spiders, one of which bites him. During a subway ride home, a fight reveals that he has developed superhuman speed, strength, the ability to stick to surfaces, and a heightened sense of danger.
In his father's documents, Peter finds an algorithm and gives it to Connors. This was the missing key to his serum. In school, Peter damages school property during a confrontation with Flash and Uncle Ben is forced to work late so that he can pick up Peter. Uncle Ben tells Peter to escort Aunt May home that night. Peter ignores Uncle Ben in favour of helping Connors test their serum on a three-legged mouse. After he returns home, Peter argues with Uncle Ben about not picking up Aunt May, and Peter leaves. At a grocery store, the clerk rudely refuses to let Peter buy a drink, and when a thief steals money from the till, Peter lets the thief escape. Uncle Ben, searching the streets for Peter, confronts the thief, but is shot and killed. Peter responds to the gunshot and finds Uncle Ben's body. After seeing a police sketch of the suspect, Peter realizes it was the thief he let go.
Peter hunts the killer, taking out other criminals as a result and attracting police attention for his vigilante actions. During one confrontation he falls into an abandoned gym, where a luchador-wrestling poster inspires him to create a mask to hide his identity. He creates a costume for himself with a spandex suit, and builds wrist-mounted mechanical devices that shoot biocable strands. Peter later reveals he is the vigilante to Gwen and they kiss.
Seeing that the test mouse has regrown its missing limb using lizard DNA, Ratha demands human trials begin immediately. Connors refuses to risk innocent lives and is fired. Desperate, Connors tests the serum on himself and his missing arm regenerates. He learns that Ratha is going to test the serum on human subjects and goes to stop him, but his body begins to mutate. By the time he finds Ratha on the Williamsburg Bridge Connors has transformed into a large Lizard creature, tossing cars, including Ratha's, over the side of the bridge. Peter, now calling himself Spider-Man, manages to save Ratha and other civilians. Connors retreats into the sewer and reverts to human form.
Connors and Peter later meet and Peter suspects he is the Lizard, and later unsuccessfully confronts Connors' Lizard form in the sewers, leaving behind his camera. Connors learns Peter's identity via the name on the camera and pursues him to Midtown Science High School where they fight. In response, the police start a manhunt for both Spider-Man and Lizard. Connors heads to Oscorp, intending to disperse his serum across the city and make everyone into lizards, while Gwen develops an antidote in Oscorp's lab. The police corner Spider-Man and Captain Stacy discovers that he is Peter, but lets him escape to go save Gwen. Spider-Man manages to replace Connors' serum with the antidote, reverting Connors to human form, but not before Connors mortally wounds Captain Stacy. Before his death, Captain Stacy makes Peter promise to stay away from Gwen for her safety. He initially keeps the promise, but later admits to Gwen that failed promises are the best kind, hinting that he will continue to see her.
In a scene after the credit roll, an imprisoned Connors is confronted by an unseen man who asks whether Connors had told Peter the truth about his father's fate. Connors swiftly replies, "No", and demands that Peter be left alone before the mysterious man disappears.
Plot recap from Spider-Man Movies Wiki
