Purpose: Cholangiocarcinoma is a fatal tumor with limited therapeutic options. We have reported that calmodulin antagonists tamoxifen and trifluoperazine induced apoptosis in cholangiocarcinoma cells. Here, we determined the effects of tamoxifen on tumorigenesis and the molecular mechanisms of tamoxifen-induced apoptosis. Experimental Design: Nude mice xenograft model of cholangiocarcinoma was used and tamoxifen was given i.p. and intratumorally. Cholangiocarcinoma cells were used to characterize molecular mechanisms of tamoxifen-induced apoptosis in vitro. Results: I.p. or intratumoral injection of tamoxifen decreased cholangiocarcinoma tumorigenesis by 40% to 80% in nude mice. In cells isolated from tumor xenografts, tamoxifen inhibited phosphorylation of AKT (pAKT) and cellular FLICE like inhibitory protein (c-FLIP). Immunohistochem-ical analysis further showed that pAKT was identified in all nontreated tumors but was absent in tamoxifen-treated tumors. In vitro, tamoxifen activated caspase-8 and caspase-10, and their respective inhibitors partially blocked tamoxifen-induced apoptosis. Overexpression of c-FLIP inhibited tamoxifen-induced apoptosis and enhanced tumorigenesis of cholangiocarcinoma cells in nude mice, whereas deletion of the calmodulin-binding domain on c-FLIP restored the sensitivity to tamoxifen and inhibited tumorigenesis. With two additional cholangiocarcinoma cell lines, we confirmed that the expression of FLIP is an important factor in mediating spontaneous and tamoxifen-induced apoptosis. Conclusions: Thus, tamoxifen inhibits cholangiocarcinoma tumorigenesis in nude mice. Tamoxifen-induced apoptosis is partially dependent on caspases, inhibition of pAKT. and FLIP expression. Further, calmodulin-FLIP binding seems to be important in FLIP-mediated resistance to tamoxifen. Therefore, the present studies support the concept that tamoxifen may be used as a therapy for cholangiocarcinoma and possibly other malignancies in which the calmodulin targets AKTand c-FLIP play important roles in the tumor pathogenesis. © 2009 American Association for Cancer Research.