Lysosomes play vital roles in the degradation and recycling of various macromolecules, protein secretion, energy metabolism, and cell signaling. Increasing evidence suggests that the deregulated lysosomes contribute to tumorigenesis and therapeutic resistance. However, its underlying mechanisms remain elusive. We herein identified transmembrane protein 9 (TMEM9) as a crucial protein modulating lysosomal metabolism and lysosome-associated signaling. The TMEM9 gene is markedly amplified in breast cancer, correlated with its transcriptional upregulation. TMEM9 depletion suppressed the proliferation of breast cancer cells highly expressing TMEM9. Consistently, Tmem9 knock-out inhibited mammary tumorigenesis in the genetically engineered mouse model. Conversely, the ectopic expression of TMEM9 in TMEM9 low breast cancer cells promoted cell proliferation. The lysosome purification and proteomics approach showed that TMEM9 physically and functionally interacts with LAMTOR4, a subunit of the Ragulator complex, to hyperactivate mTOR signaling in breast cancer. Moreover, the pharmacological blockade of the TMEM9-v-ATPase axis combined with mTOR inhibitors exhibited the synergistic growth inhibition of breast cancer cells. Our results reveal the mechanism of the TMEM9-v-ATPase-activated mTOR signaling and further propose the TMEM9-v-ATPase axis as a therapeutic target overcoming mTOR inhibitor resistance of breast cancer.