Cancer cells have developed chemoresistance and have improved their survival through the upregulation of autophagic mechanisms that protect mitochondrial function. Here, we report that the traditional Chinese anticancer agent tubeimoside I (Tub), which is a potent inhibitor of autophagy, can promote mitochondria-associated apoptosis in lung cancer cells. We found that Tub disrupted both mitochondrial and lysosomal pathways. One of its mechanisms was the induction of DRP1-mediated mitochondrial fragmentation. Another mechanism was the blocking of late-stage autophagic flux via impairment of lysosomal acidification through V-ATPase inhibition; this blocks the removal of dysfunctional mitochondria and results in reactive oxygen species (ROS) accumulation. Excessive ROS accumulation causes damage to lysosomal membranes and increases lysosomal membrane permeability, which leads to the leakage of cathepsin B. Finally, cathepsin B upregulates Bax-mediated mitochondrial outer membrane permeability and, subsequently, cytosolic cytochrome C-mediated caspase-dependent apoptosis. Thus, the cancer cell killing effect of Tub is enhanced through the formation of a positive feedback loop. The killing effect of Tub on lung cancer cells was verified in xenografted mice. In summary, Tub exerts a dual anticancer effect that involves the disruption of mitochondrial and lysosomal pathways and their interaction and, thereby, has a specific and enhanced killing effect on lung cancer cells.