On the basis of previous findings that certain lung carcinoma cell lines are resistant to the dual PI3K/mTOR inhibitor PI103, we searched for new strategies to overcome this resistance. Here, we report that the lysosomotropic agent chloroquine (CQ) reverses the resistance of lung carcinoma cells to PI3K/mTOR inhibition and primes cells for PI103-induced apoptosis. Investigations of the underlying mechanism of this cooperative interaction show that PI103 increases lysosomal volume and function, as indicated by upregulation of the lysosomal marker protein LAMP-1 and maturation of the lysosomal enzyme cathepsin B, whereas CQ destabilizes lysosomal membranes. Together, CQ and PI103 act in concert to trigger lysosomal membrane permeabilization, resulting in the activation of caspases and apoptosis. The broad-range caspase inhibitor zVAD.fmk significantly decreases PI103-induced and CQ-induced loss of cell viability, indicating that caspases are required for cell death induction. Importantly, inhibition of lysosomal enzymes by CA-074me significantly reduces PI103-mediated and CQ-mediated loss of cell viability, showing that lysosomal enzymes are critical mediators of PI103/CQ-induced cytotoxicity. In conclusion, CQ overcomes resistance of lung carcinoma cells to PI103-induced apoptosis by cooperating with PI103 to trigger lysosome-mediated apoptosis. These findings have important implications for developing effective PI3K/mTOR inhibitor-based therapies for lung cancer.