Non-small cell lung carcinomas (NSCLCs) are typically resistant against apoptosis induced by standard chemotherapy. We evaluated the effects of the two potential antitumor agents of the lamellarin class on a highly apoptosis-resistant NSCLC cell line. Both the marine alkaloid lamellarin-D and its synthetic amino derivative PM031379 induced the activation of Bax, the mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF), as well as the activation of caspase-3. However, only PM031379 triggered cell death and sign of nuclear apoptosis coupled to the nuclear translocation of AIF. Depletion of AIF with small interfering RNA or microinjection of a neutralizing anti-AIF antibody largely prevented PM031379-induced cytotoxicity, underscoring the essential contribution of AIF to NSCLC killing. Using NSCLC cells lacking mitochondrial DNA, we showed that the generation of mitochondrial reactive oxygen species (ROS) was crucial for the PM031379-induced translocation of AIF to the nucleus and subsequently cell death. Pretreatment of NSCLC cells with menadione, a mitochondrial ROS generator, was able to restore the deficient chemotherapy-induced apoptosis of NSCLC cells. Altogether, these data suggest that mitochondrial ROS generation is crucial for overriding the chemoresistance of NSCLC cells. Moreover, this study delineates the unique mechanism of action of lamellarins as potential anticancer agents.