RUNX3 is frequently inactivated by DNA hypermethylation in numerous cancers. Here, we show that RUNX3 has an important role in modulating apoptosis in immediate response to tumor necrosis factor-related apoptosis-including ligand (TRAIL). Importantly, no combined effect of TRAIL and RUNX3 was observed in non-cancerous cells. We investigated the expression of the death receptors (DRs) DR4 and DR5, which are related to TRAIL resistance. Overexpression of RUNX3 increased DR5 expression via induction of the reactive oxygen species (ROS)-endoplasmic reticulum (ER) stress-effector CHOP. Reduction of DR5 markedly decreased apoptosis enhanced by the combined therapy of TRAIL and RUNX3. Interestingly, RUNX3 induced reactive oxygen species production by inhibiting SOD3 transcription via binding to the Superoxide dismutase 3 (SOD3) promoter. Additionally, the combined effect of TRAIL and RUNX3 decreased tumor growth in xenograft models. Our results demonstrate a direct role for RUNX3 in TRAIL-induced apoptosis via activation of DR5 and provide further support for RUNX3 as an anti-tumor.