Background: Cholangiocarcinoma originates from bile duct epithelial cells in the intrahepatic and extrahepatic biliary system. We recently observed that triptolide (a diterpenoid triepoxide) is effective in inducing apoptosis in pancreatic tumors. Death receptors 4 and 5 are overexpressed in several cancer types, and their activation by tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell death. The principal objective of this study was to determine the effects of combination therapy with TRAIL and triptolide in cholangiocarcinoma. Materials and Methods: Two cholangiocarcinoma cell lines were incubated with various doses of triptolide and TRAIL, alone and in combination; cell viability was assessed at 24 and 48 h. Annexin-V staining and caspase-3 activity were measured after 24 h of triptolide, TRAIL or combination treatment. Western blots assessed protein levels of poly(ADP-ribose) polymerase (PARP) and X-linked inhibitor of apoptosis (XIAP). Results: Combination treatment using TRAIL and triptolide decreased cell viability in all cell lines at 48 h, with greater cell killing than that which was observed with either drug alone. This decrease in viability was associated with increases in annexin-V staining and caspase-3 activity. Western blot analysis demonstrated increases in PARP cleavage and decreases in XIAP expression that were dose-dependent. Conclusions: TRAIL and triptolide in combination decreased cell viability and enhanced apoptosis. Furthermore, Western blot analysis suggests that triptolide sensitizes cells to TRAIL-induced apoptotic cell death by inhibiting expression of XIAP, a protein known to inhibit apoptosis. Our results demonstrate that combination of TRAIL and triptolide enhance apoptosis in cholangiocarcinoma cell lines.