Prostate cancer is a major health threat for American men. Therefore, the development of effective therapeutic options is an urgent issue for prostate cancer treatment. In this study, we evaluated the effect of glycogen synthase kinase-3β (GSK-3β) suppression on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human prostate cancer cell lines. In the presence of lithium chloride (LiCl) or SB216763, the GSK-3β inhibitors, TRAIL-induced cell death was dramatically enhanced, and the enhanced cell death was an augmented apoptotic response evidenced by increased Annexin V labeling and caspase-3 activation. GSK-3β gene silencing mediated by a small interference RNA (siRNA) duplex also sensitized the cells to TRAIL, confirming the specificity of GSK-3β suppression. Importantly, TRAIL stimulation increased GSK-3β tyrosine phosphorylation at Y216, suggesting that GSK-3β is activated by TRAIL. Furthermore, TRAIL sensitization was associated with increased proteolytic procession of caspase-8 and its downstream target BID, and z-IETD-FMK, the inhibitor specific to active caspase-8 totally blocked LiCl-induced TRAIL sensitization. Finally, Trichodion, a potent nuclear factor-κB (NF-κB) inhibitor, could not affect LiCl-induced TRAIL sensitization, although GSK-3β inhibitors significantly blocked TRAIL-reduced NF-κB activity in prostate cancer cells. These results indicate that GSK-3β suppression sensitizes prostate cancer cells to TRAIL-induced apoptosis that is dependent on caspase-8 activities but independent of NF-κB activation, and suggest that a mechanism involving GSK-3β activation may be responsible for TRAIL resistance in prostate cancer cells.