"Evangelion: 3.0+1.0: Thrice Upon A Time" serves as the final installment in the Rebuild of Evangelion film series, a reimagining of the original "Neon Genesis Evangelion" anime. The story takes place after the events of "Evangelion: 3.0 You Can (Not) Redo," where the world has been devastated by the Near-Third Impact. Shinji Ikari, the protagonist, remains emotionally scarred and guilt-ridden following his role in the catastrophic event. The narrative begins with Shinji and his companions, including Asuka Shikinami Langley and Rei Ayanami, residing in a temporary village called Village 3, a rare haven of normalcy amidst the post-apocalyptic landscape.

The village is overseen by Misato Katsuragi and her organization, WILLE, which continues its fight against NERV and the mysterious entity known as the Evangelion Mark.06. As Shinji struggles to reconcile his past actions and find purpose, he encounters familiar faces such as Kaworu Nagisa, who offers him guidance, and Gendo Ikari, his estranged father, who remains determined to complete the Human Instrumentality Project. The project aims to merge all human consciousness into a single entity, a goal that has driven much of the series' conflict.

The plot intensifies as WILLE launches a final assault on NERV headquarters to prevent the Fourth Impact, a cataclysmic event that would bring about the end of humanity. Shinji, after confronting his inner turmoil and gaining clarity, decides to confront Gendo directly. The father-son confrontation reveals Gendo's motivations, rooted in his grief over the loss of his wife, Yui Ikari, and his desire to reunite with her through the Instrumentality Project. Shinji, however, rejects this path, choosing instead to forge a future where individuals can coexist despite their pain and differences.

The climax involves a metaphysical battle within the Anti-Universe, a realm beyond conventional space and time, where Shinji and Gendo's ideologies clash. Shinji ultimately prevails, using the power of the Evangelions to rewrite reality and create a world free from the cycle of destruction and suffering. The film concludes with Shinji and his friends emerging into a new, hopeful reality, symbolizing a fresh start for humanity.

Key characters include Shinji Ikari, whose journey of self-acceptance and growth forms the emotional core of the story; Asuka Shikinami Langley, a skilled pilot grappling with her own vulnerabilities; Rei Ayanami, whose evolving humanity contrasts with her origins as a clone; and Gendo Ikari, whose obsession with reuniting with Yui drives much of the conflict. The film ties together the series' themes of identity, connection, and the human condition, providing a definitive conclusion to the Rebuild saga.