Under constant attack by Angels, NERV introduces two new pilots: the mysterious Makinami Mari Illustrous and the intense Asuka Langley Shikinami. Parallel to the incursion, Gendo Ikari and SEELE proceed the secret project that involves both Rei and Shinji. The film continues the story of Evangelion: 1.0 You Are (Not) Alone, with Shinji Ikari continuing his role as a pilot of one of the gigantic "Evangelion" mecha as part of NERV's ongoing fight against the mysterious creatures known as Angels. While replicating many scenes and plot elements from the original series, the film also introduces new ones, including newly designed creatures and new characters, such as Mari Illustrious Makinami, and integrates newly available 3D CG technology. Its ending paves the way for the significant storyline departures from the original series in Evangelion: 3.0 You Can (Not) Redo.

The fate of the world is threatened by seemingly monstrous entities known as Angels. NERV is an organisation set up to counter this threat and it is up to young pilots to protect Earth but exactly what are the real motives behind NERV? The film focuses on a young teenager named Shinji Ikari, who is asked to pilot a mecha known as "Evangelion Unit-01" to protect the world from mysterious creatures known as Angels. The plot is largely a point-for-point adaptation of the first six episodes of the original anime. While some scenes and events are replications of the original series, others unfold differently with new or omitted scenes and newly-available 3D CG technology. The film received a positive response from fans, with Hideaki Anno himself calling it a "faithful remake of the original series". The film was ranked as the 4th-highest grossing anime film at the Japanese box office during 2007, earning a total of 2 billion yen.