Aim: To investigate the antiproliferative activity and apoptosis-inducing effects of bufalin on human osteosarcoma cell lines. Methods: U-2OS and U-2OS meth-otrexate (MTX) 300-resistant cell lines were treated with bufalin. Cell viability was assessed using the MTT assay. Cell-cycle status, apoptosis-inducing effects,and the expression of apoptosis-related proteins were evaluated by flow cytometry,fluorescent staining, DNA fragmentation assays, and Weste blotting. The ef-fect of bufalin on dihydrofolate reductase (DHFR) expression was studied by RT-PCR and Weste blotting. Results: Bufalin inhibited cell growth in both U-2OS and U-2OS MTX300 cells. The induction of G2/M cell-cycle arrest was also seen in the cells treated with bufalin. The induction of apoptosis by bufalin was con-firmed by increased expression of the tumor suppressor protein p53 and the in-creased ratio of the Bax/Bcl-2 proteins. Bufalin induced apoptosis to the same extent in both cell lines without regard to DHFR levels in the cells. Conclusion:Bufalin inhibited the growth of and induced apoptosis in both MTX-sensitive and MTX-resistant human osteosarcoma U-2OS cells. The apoptosis-inducing effect of bufalin was not influenced by the presence of high levels of the DHFR protein.