Capsaicin, a pungent molecular compound present in many hot peppers, exerts anticancer activities against various human cancer cell lines by inducing apoptosis. However, the effects of capsaicin on human osteosarcoma (OS) as well as the related mechanisms remain to be fully elucidated. In the present study, the anticancer effects of capsaicin on 3 human OS cell lines (MG63, 143B and HOS) were investigated. Various concentrations of capsaicin (50-300 µM) effectively decreased cell viability in all 3 OS cell lines in a dose-dependent manner. Notably, capsaicin-induced apoptosis was observed when OS cells were treated with relatively high concentrations of capsaicin (starting at 250 µM). In addition, the mitochondrial apoptotic pathway was involved in the capsaicin-induced apoptosis in the OS cells. Meanwhile, our results also indicated that at relatively low concentrations (e.g., 100 µM), capsaicin could inhibit the proliferation, decrease the colony forming ability and induce G0/G1 phase cell cycle arrest of OS cells in a dose-dependent manner. Moreover, our results revealed that the anticancer effects induced by capsaicin on OS cell lines involved multiple MAPK signaling pathways as indicated by inactivation of the ERK1/2 and p38 pathways and activation of the JNK pathway. Furthermore, the results of animal experiments showed that capsaicin inhibited tumor growth in a xenograft model of human OS. In conclusion, these results indicate that capsaicin may exert therapeutic benefits as an adjunct to current cancer therapies but not as an independent anticancer agent.