Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy; however, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Resistance to apoptosis may unmask TRAIL signaling cascades favoring tumor biology. Thus, our aim was to examine whether TRAIL is expressed by human cholangiocarcinomas, and if so, does it promote a malignant phenotype. To address this objective, TRAIL expression in human liver specimens was evaluated by immunohistochemistry. The effect of TRAIL on tumor cell migration, invasion, and proliferation was examined in three human cholangiocarcinoma cell lines. TRAIL expression was up-regulated by cholangiocytes in the preneoplastic disease, primary sclerosing cholangitis, and in human cholangiocarcinoma specimens. TRAIL promoted tumor cell migration and invasion, but did not induce cell proliferation. TRAIL-mediated cell migration and invasion was nuclear factor-B (NF-B) dependent. These data demonstrate that TRAIL promotes cell migration and invasion via a NF-B-dependent pathway in human cholangiocarcinoma cell lines; an observation which has a potential negative implication for TRAIL in cancer therapy.