Downregulation of apoptotic signal pathway and activation of protective autophagy mainly contribute to the chemoresistance of tumor cells. Therefore, exploring efficient chemotherapeutic agents or isolating novel natural products that can trigger nonapoptotic and nonautophagic cell death such as lysosome-associated death is emergently required. We have recently extracted a saponin, gypenoside L (Gyp-L), from Gynostemma pentaphyllum and showed that Gyp-L was able to induce nonapoptotic cell death of esophageal cancer cells associated with lysosome swelling. However, contributions of vacuolization and lysosome to cell death remain unclear. Herein, we reveal a critical role for NADPH oxidase NOX2-mediated vacuolization and transcription factor EB (TFEB) activation in lysosome-associated cell death. We found that Gyp-L initially induced the abnormal enlarged and alkalized vacuoles, which were derived from lipid rafts dependent endocytosis. Besides, NOX2 was activated to promote vacuolization and mTORC1-independent TFEB-mediated lysosome biogenesis. Finally, raising lysosome pH could enhance Gyp-L induced cell death. These findings suggest a protective role of NOX2-TFEB-mediated lysosome biogenesis in cancer drug resistance and the tight interaction between lipid rafts and vacuolization. In addition, Gyp-L can be utilized as an alternative option to overcome drug-resistance though inducing lysosome associated cell death.