Gallbladder carcinoma (GBC) is an aggressive malignancy with high mortality, mainly due to the reduced chance of curative resection and the resistance to chemotherapeutic drugs. Here, we showed that cellular Fas-associated death domain-like interleukin-1 converting enzyme inhibitory protein (c-FLIP), an anti-apoptotic protein, was over-expressed in the most of gallbladder carcinoma tissues, as judged by immunohistochemistry. Semi-quantitation was performed by determining the percentage of c-FLIP-positive cells: no positive cells (-), approximately 1% positive cells (+), approximately 30% positive cells (++), and >70% positive cells (+++). Out of the 35 tissue specimens of gallbladder carcinoma, positive c-FLIP expression was found in 26 samples (6/positive+++, 13/++, 7/+), whereas negative or weak c-FLIP staining was detected in normal (1/+, 9/-) and adenomatous (2/+, 8/-) gallbladder tissues. Then, we used a small interference RNA (siRNA), which can substantially down-regulate the expression levels of c-FLIP mRNA and protein in GBC-SD and SGC-996 human gallbladder carcinoma cells, as confirmed by real-time PCR and western blot analyses. Furthermore, the combined treatment with the c-FLIP siRNA and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) significantly induced apoptosis in gallbladder carcinoma cells, as judged by the increases in pyknosis, caspase-3/7 activities, and Annexin V-propidium iodide labeling, a marker for chromatin condensation. Thus, the siRNA-mediated down-regulation of c-FLIP profoundly enhances the sensitivity to TRAIL-induced apoptosis. In conclusion, c-FLIP expression is up-regulated in gallbladder carcinoma and the down-regulation of c-FLIP sensitizes TRAIL-induced apoptosis. The present study provides a potent strategy for the treatment of gallbladder carcinoma by targeting the c-FLIP.