As a young boy, Peter Parker (Andrew Garfield) experiences a traumatic event when he discovers his father Richard's study has been ransacked. His father quickly gathers concealed documents before Peter's parents take him to live with his Aunt May (Sally Field) and Uncle Ben (Martin Sheen). Unfortunately, Peter's parents, particularly his mother Mary ([Embeth Davidtz]), are later killed in a tragic plane crash.
Years later, as a high school student at Midtown Science High School, Peter faces bullying from Flash Thompson ([Chris Zylka]), while simultaneously harboring romantic feelings for the stunning Gwen Stacy (Emma Stone), who happens to be the daughter of police captain George Stacy ([Denis Leary]). With a strong passion for photography, Peter is often seen carrying his camera, capturing moments.
While exploring his father's past, Peter unearths some of his hidden documents and discovers that his father had been collaborating with scientist Dr. Curt Connors (Rhys Ifans) at Oscorp, working on a project that merges human and animal DNA in an effort to cure diseases. Curiously, following Richard's passing, Connors had never reached out to the Parker family.
Driven by curiosity and ambition, Peter disguises himself as one of the interns during a new induction at Oscorp to meet Connors. However, he must tread carefully as Gwen is already the senior intern under Connors. After Peter cleverly answers a question posed by Connors, Gwen, realizing who he is, opts not to expose him.
Meanwhile, Connors grapples with pressure from his superior, Dr. Ratha ([Irrfan Khan]), who pushes him to complete the serum designed to cure the terminally ill Norman Osborn. Connors has personal stakes as well, wishing to use the serum to restore his lost arm.
In a pivotal moment, Peter sneaks into a lab and is bitten by a genetically modified spider, leading to extraordinary changes in his physical abilities—enhancing his strength and speed while granting him a sixth sense known as a "spider-sense." Soon after, he also discovers an algorithm in his father's documents known as the 0 0 decay rate algorithm, which he shares with Connors, believing it to be the crucial missing piece to their project.
However, as tensions build, Peter becomes estranged from Uncle Ben, especially after he opts to visit Connors instead of escorting Aunt May. This decision costs him dearly when a chase ensues at a grocery store, leading to the loss of Ben's life at the hands of a thief—an assailant Peter had chosen to let go earlier.
In his quest for vengeance and justice, Peter begins adopting a vigilante lifestyle, which earns him the moniker Spider-Man. After creating a unique costume and designing mechanical devices that help him shoot high-strength webbing, he forges a bond with Gwen—whom he soon reveals his true identity to.
As Captain Stacy leads the efforts to contain Spider-Man's actions, Connors' experiments take a sinister turn. Ignoring ethics, Connors injects himself with the serum, resulting in a transformation into a monstrous Lizard creature. The narrative escalates as Connors seeks to unleash his serum across the city to turn everyone into lizards, while Gwen works tirelessly to create an antidote.
A dramatic showdown unfolds at Oscorp, where Spider-Man races against time to thwart Connors' plans and save Gwen. The climax sees Captain Stacy becoming a casualty, urging Peter to keep his distance from Gwen for her safety before he succumbs to his injuries.
As the story resolves, a post-credits scene teases future intrigues when Connors, now imprisoned, encounters a mysterious figure who questions him regarding Peter's father. Connors' response, “No,” leaves audiences pondering the deeper secrets surrounding Peter's origins and his father's past affiliations.
