Sulforaphane enhances TRAIL-induced apoptosis through the induction of DR5 expression in human osteosarcoma cells. Sulforaphane (SFN), a naturally occurring isothiocyanate, is an attractive agent because of its potent anticancer effects. SFN suppresses the proliferation of various cancer cells in vitro and in vivo. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also one of the most promising candidates for cancer therapeutics owing to its ability to selectively induce apoptosis in tumor cells. In this study, we report that SFN enhances TRAIL-induced apoptosis in human osteosarcoma cells, Saos2 and MG63. The apoptosis induced by co-treatment with SFN and TRAIL was markedly blocked by a dominant negative form of the TRAIL receptor or caspase inhibitors. The combined use of SFN and TRAIL effectively induced Bid cleavage and the activation of caspases 8, 10, 9 and 3 at ineffective concentrations for each agent. SFN upregulated the expression of death receptor 5 (DR5), a receptor for TRAIL, at mRNA and protein levels in a dose-dependent manner. In addition, the SFN-mediated sensitization to TRAIL was reduced by DR5 siRNA, suggesting that the sensitization was at least partially mediated through the induction of DR5 expression. Furthermore, SFN sensitized TRAIL-induced apoptosis in a p53-independent manner. On the other hand, SFN neither induced DR5 protein expression or enhanced TRAIL-induced apoptosis in normal human peripheral blood mononuclear cells. Thus, combined treatment with SFN and TRAIL might be a promising therapy for osteosarcoma.