DNA mismatch repair (MMR) is essential for maintaining genome integrity, yet its regulation by non-protein biomolecules remains poorly understood. Here, we uncover nuclear phosphatidylinositol 3-phosphate (PI3P) as a previously unrecognized lipid mediator of MMR. PI3P, canonically produced by the Beclin-1/Vps34 complex in the cytoplasm, is also synthesized within the nucleus, where it plays a direct and functionally critical role in orchestrating MMR. Nuclear PI3P physically associates with core MMR proteins and facilitates the assembly of MutSα and MutSβ complexes, enabling mismatch recognition and subsequent repair. Inhibition of Beclin-1 or Vps34 compromises nuclear PI3P production, which impairs MMR recognition complex formation, leading to microsatellite instability. Conversely, supplementation with exogenous PI3P restores MMR recognition complex assembly and mismatched substrate binding in vitro, confirming its functional necessity. Our study reveals a noncanonical, autophagy-independent function of the nuclear Beclin-1/Vps34 complex in genome surveillance, and identifies nuclear phospholipids as versatile regulators of DNA repair.