BIX-01294 (BIX), a G9a histone methyltransferase inhibitor, has been reported for its anti-proliferative and anticancer activities against various cancer cell lines. In this study, we investigated whether BIX could sensitize TRAIL-mediated apoptosis in various cancer cells. Combined treatment with BIX and TRAIL markedly induced apoptosis in human renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MCF-7), and lung carcinoma (A549) cells. In contrast, BIX and TRAIL co-treatment did not induce apoptosis in normal cells, specifically mouse kidney cell (TCMK-1) and human skin fibroblast (HSF). BIX downregulated protein expression levels of XIAP and survivin at the post-translational level. Overexpression of survivin markedly blocked combined BIX and TRAIL treatment-induced apoptosis, but XIAP had no effect. Furthermore, BIX induced upregulation of DR5 expression at the transcriptional levels, and knockdown of DR5 expression using small interfering RNAs (siRNAs) markedly attenuated BIX and TRAIL-induced apoptosis. Interestingly, siRNA-mediated G9a histone methyltransferase knockdown also enhanced TRAIL-induced apoptosis in Caki cells. However, knockdown of G9a did not change expression levels of XIAP, survivin, and DR5. Therefore, BIX-mediated TRAIL sensitization was independent of histone methyltransferase G9a activity. Taken together, these results suggest that BIX facilitates TRAIL-mediated apoptosis via downregulation of survivin and upregulation of DR5 expression in renal carcinoma Caki cells. ▶ BIX facilitates TRAIL-mediated apoptosis in human renal carcinoma Caki cells. ▶ Downregulation of survivin contributes to BIX plus TRAIL-induced apoptosis. ▶ Upregulation of DR5 is involved in BIX plus TRAIL-mediated apoptosis. ▶ BIX-mediated TRAIL sensitization is independent of ROS production.