Lysosomal localization of mammalian target of rapamycin complex 1 (mTORC1) is a critical step for activation of the molecule. Rag GTPases are essential for this translocation. Here, we demonstrate that Nudix-type motif 2 (NUDT2) is a novel positive regulator of mTORC1 activation. Activation of mTORC1 is impaired in NUDT2-silenced cells. Mechanistically, NUDT2 binds to Rag GTPase and controls mTORC1 translocation to the lysosomal membrane. Furthermore, NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells, as NUDT2-silenced cells arrest in G0/G1 phases. Taken together, these results show that NUDT2 is a novel complex formation enhancing factor regulating mTORC1-Rag GTPase signaling that is crucial for cell growth control. Display omitted •NUDT2 is identified as a novel regulator of Rag GTPase-mTORC1 signaling.•NUDT2 was associated with recruitment of mTORC1 to the lysosomal membrane.•The physical interaction between NUDT2 and Rag GTPase heterodimers is required for the lysosomal localization of mTORC1.•NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells.