Methylene blue (MB) is a widely studied agent currently under investigation for its properties relating to photodynamic therapy (PDT). Recent studies have demonstrated that MB exhibits profound phototoxicity affecting a variety of tumor cell lines. However, the mechanistic explanation for methylene-blue-mediated photodynamic therapy (MB-PDT) in the context of melanoma therapy is still obscure. In the present study, B16F1 melanoma cells were treated by MB-PDT under different conditions, and thereafter subjected to cell viability detection assays. MB-PDT could induce intense apoptotic cell death through a series of steps beginning with the photochemical generation of reactive oxygen species that activate the caspase-9/caspase-3 apoptosis pathway. Blocking activation of caspase-3 and induction of oxidative stress by caspase inhibitor and by glutathione, respectively, markedly reduced apoptotic cell death in vitro. Importantly, proteomics study defining altered protein expression in treated cells suggests the involvement of several mitochondrial proteins playing important roles in electron transfer chain, implying mitochondrial dysfunction during the treatment. Furthermore, a transplantable mouse melanoma model was utilized to estimate the effectiveness of MB-PDT in vivo. The treated mice displayed decreased tumor size and prolonged survival days, which was associated with enhanced apoptotic cell death. These results, offering solid evidence of the induction of mitochondria-related apoptosis in tumor cells, reveal new aspects of MB-PDT having potential to be a palliative treatment of melanoma.