Abstract. Bufalin has been shown to induce apoptosis in osteosarcoma cells; however, the underlying mechanism has not been elucidated. The purpose of the present study was to investigate whether mitochondria-mediated signaling pathways trigger the process of apoptosis in the U-2OS osteosarcoma cell line. Bufalin inhibited the proliferation and induced apoptosis in U-2OS cells in a time-and dose-dependent manner. Bufalin-induced apoptosis was accompanied with a significant reduction of the mitochondrial membrane potential, release of mitochondrial cytochrome c into the cytosol, activation of caspase-3, caspase-9 and poly(adenosine diphosphate ribose) polymerase, as well as downregulation of B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition pore, attenuated bufalin-induced apoptosis. In conclusion, the present study revealed that bufalin induced apoptosis in the U-2OS human osteosarcoma cell line via triggering of the mitochondrial pathway.