Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various types of malignant cancer cells, but several cancers have acquired potent resistance to TRAIL-induced cell death by unknown mechanisms. Camptothecin (CPT) is a quinolone alkaloid that induces cytotoxicity in a variety of cancer cell lines. However, it is not known whether CPT triggers TRAIL-induced cell death. In this study, we found that combined treatment with subtoxic doses of CPT and TRAIL (CPT-TRAIL) potentially enhanced apoptosis in a caspase-dependent manner. CPT-TRAIL effectively induced the expression of death receptor (DR) 5, which is a specific receptor of TRAIL, and treatment with a chimeric blocking antibody for DR5 reduced CPT-TRAIL-induced cell death, indicating that CPT functionally triggers DR5-mediated cell death in response to TRAIL. CPT-induced generation of reactive oxygen species (ROS) also preceded the upregulation of DR5 in response to TRAIL. The involvement of ROS in DR5 upregulation confirmed that pretreatment with antioxidants, including N-acetyl-L-cysteine and glutathione, significantly inhibits CPT-TRAIL-induced cell death by suppressing DR5 expression. The specific inhibitors of ERK and p38 also decreased CPT-TRAIL-induced cell death by blocking DR5 expression. In conclusion, our results suggest that CPT sensitizes cancer cells to TRAIL-mediated apoptosis via ROS and ERK/p38-dependent DR5 upregulation.