Research on triptolide, a diterpenoid epoxide found in the Thunder God Vine Tripterygium wilfordii, has increased our knowledge of the pharmacology, pharmacokinetics, toxicology and clinical application of this agent. In the present study, we aimed to identify the effects of triptolide on the apoptosis of osteosarcoma cells and to evaluate the anti-proliferative action of this agent. MG-63 cells were treated either with various doses of triptolide (50, 100 or 200 nM) or DMSO for 6, 12 and 24 h. Treatment with triptolide effectively suppressed the cell viability and induced the apoptosis of osteosarcoma MG-63 cells as detected by MTT assay and flow cytometry, respectively. In addition, by using caspase-3, caspase-8 and caspase-9 activity assays and western blot analysis, the anticancer effects of triptolide against osteosarcoma growth were found to involve activation of the DR-5/p53/Bax/caspase-9/ caspase-3 signaling pathway and the DR-5/FADD/caspase-8/lysosomal/cathepsin B/caspase-3 signaling pathway in the MG-63 cells. An important factor in the anticancer effects of triptolide against osteosarcoma was TRAIL-DR-5. The data suggest that triptolide may be a potential novel chemotherapeutic agent for osteosarcoma and acts through the TRAIL-DR-5 signaling pathway.