YM155, a small-molecule survivin inhibitor, has been reported for its anti-cancer activity in various cancer cells. In this study, we investigated the effect of YM155 to enhance TRAIL-mediated apoptosis in human renal carcinoma cells. We found that YM155 alone had no effect on apoptosis, however, combined treatment with YM155 and TRAIL markedly induced apoptosis in human renal carcinoma cells (Caki, ACHN, and A498), breast cancer cells (MDA-MB231), and glioma cells (U251MG), but not normal cells [mesangial cell (MC) and human skin fibroblast (HSF)]. YM155 induced down-regulation of Mcl-1 expression at the post-translational levels, and the overexpression of Mcl-1 markedly inhibited YM155 plus TRAIL-induced apoptosis. Furthermore, YM155 induced down-regulation of c-FLIP mRNA expression through inhibition of NF-κB transcriptional activity. Ectopic expression of c-FLIP markedly blocked YM155-induced TRAIL sensitization. Taken together, our results suggested that YM155 sensitizes TRAIL-mediated apoptosis via down-regulation of Mcl-1 and c-FLIP expression in renal carcinoma Caki cells.