Continued smoking causes tumor progression and resistance to therapy in lung cancer. Carcinogens possess the ability to block apoptosis, thus may induce development of cancers and resistance to therapy. Tobacco carcinogens have been studied widely; however, little is known about the agents that inhibit apoptosis, such as nicotine. We determine whether mitochondrial signaling mediates antiapoptotic effects of nicotine in lung cancer. The A549 cells were exposed to nicotine (1µM) followed by cisplatin (35µM) plus etoposide (20µM) for 24h. We found that nicotine prevented chemotherapy-induced apoptosis, improved cell survival and caused modest increase in DNA synthesis. Inhibition of MAPK and AKT prevented the antiapoptotic effects of nicotine and decreased chemotherapy-induced apoptosis. siRNAMEK1 blocked antiapoptotic effects of nicotine, while, siRNAMEK2 blocked chemotherapy-induced apoptosis. Nicotine prevented chemotherapy-induced reduction in mitochondrial membrane potential and caspase-9 activation. Antiapoptotic effects of nicotine were blocked by mitochondrial anion channel inhibitor, DIDS. Chemotherapy enhanced translocation of proapoptotic Bax to the mitochondria; while, nicotine blocked these effects. Nicotine upregulated AKT mediated antiapoptotic XIAP and phosphorylated proapoptotic Bad. The A549-rho0 cells that lack mitochondrial DNA demonstrated partial resistance to chemotherapy-induced apoptosis; however, blocked the antiapoptotic effects of nicotine. Accordingly, we provide evidence that nicotine modulates mitochondrial signaling and inhibits chemotherapy-induced apoptosis in lung cancer. The mitochondrial regulation of nicotine imposes an important mechanism that can critically impair the treatment of lung cancer since many cancer therapeutic agents induce apoptosis via the mitochondrial death pathway. Strategies armed at understanding nicotine-mediated signaling may facilitate the development of improved therapies in lung cancer.