Cisplatin is an anticancer drug that can induce apoptosis. In this study, we investigated the effect of mitochondrial DNA (mtDNA) depletion on cisplatin-induced cell death using a human osteosarcoma cell line (143B) and mtDNA-depleted 143B cells (143B-rho0). Results showed that cisplatin decreased cell survival in 143B-rho0 cells. Moreover, cisplatin induced a greater extent of apoptosis-associated DNA fragmentation and caspase 3 activation in 143B-rho0 cells. The release of mitochondrial cytochrome c into cytosol by cisplatin was enhanced more obviously in 143B cells than in 143B-rho0 cells; however, in the control group of 143B-rho0 cells, it was already dramatically greater. Depletion of mtDNA may increase sensitivity of cells to cisplatin-induced apoptosis by enhancing caspase 3 activation via both cytochrome c-dependent and -independent pathways.