Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a cytokine that selectively induces apoptosis in many tumor cells while leaving normal cells intact and is thus an attractive candidate for antitumor therapies. This paper reports that the combination of tunicamycin plus TRAIL produced a strong synergistic effect in TRAIL-sensitive human colon cancer HCT116 cells and TRAIL-resistant HT-29 cells. On a cellular mechanistic level, tunicamycin-enhanced TRAIL-induced apoptosis by death receptor (DR) 5 upregulation and DR4 deglycosylation. Knockdown of DR5 but not DR4 expression by specific shRNAs or siRNAs significantly increased tunicamycin-mediated and TRAIL-mediated cell viability. DR5 induction was regulated by C/EBP homologous protein (CHOP) and JNK as CHOP siRNA or JNK inhibitor SP600125 considerably abolished the DR5 induction. In addition, tunicamycin inhibited epidermal growth factor receptor glycosylation and the downstream signaling pathways, Akt and extracellular signal-regulated kinases activation, which might also be required for TRAIL sensitization by tunicamycin. In summary, tunicamycin effectively enhanced TRAIL-induced apoptosis might through JNK-CHOP-mediated DR5 upregulation and the inhibition of the epidermal growth factor receptor pathway.