Abstract: Melatonin (N-acetyl-5-methoxytryptamine) has differentiated the effects on apoptosis in normal and cancer cells. The mechanisms that account for the opposite effects on these cells are not adequately understood. In this study, we investigated the combined effect of melatonin and thapsigargin (TG) on apoptosis of renal cancer cells. Cotreatment with melatonin (1 mm) and TG (50 nm) induced approximately 10-fold expression levels of CCAAT-enhancer-binding proteins homologous protein (CHOP) compared with that of TG (50 nm) alone. Downregulation of CHOP expression using small interfering RNAs markedly attenuated melatonin plus TG-mediated apoptosis. In addition, cotreatment with TG- and melatonin-induced CHOP upregulation likely relates to melatonin’s antioxidant capacity because we proved that this CHOP upregulation is melatonin receptor independent. Our results collectively demonstrate that the upregulation of CHOP contributes to the enhancing effect of melatonin plus TG on apoptosis in cancer cells.