In the past decade, the incidence and mortality rates of cholangiocarcinoma (CCA) have been increasingworldwide. The relatively low responsiveness of CCA to conventional chemotherapy leads to poor overall survival.Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has emerged as the mostpromising anti-cancer therapeutic agent since it is able to selectively induce apoptosis of tumor cells but notnormal cells. In this study, we aimed to investigate the therapeutic effect of TRAIL in CCA cell lines (M213,M214 and KKU100) compared with the immortal biliary cell line, MMNK1, either alone or in combination witha subtoxic dose of 5-fluorouracil (5-FU). We found that recombinant human TRAIL (rhTRAIL) was a potentialagent which significantly inhibited cell proliferation and mediated caspase activities (caspases 8, 9 and 3/7) andapoptosis of CCA cells. The combined treatment of rhTRAIL and 5-FU effectively enhanced inhibition of CCAcell growth with a smaller effect on MMNK1. Our finding suggests TRAIL to be a novel anti-cancer therapeuticagent and advantage of its combination with a conventional chemotherapeutic drug for effective treatment ofCCA.