Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family of cytokines, causes apoptosis by caspase activation in various cell types, particularly in transformed cells. Numerous types of tumors are relatively resistant to TRAIL-induced cytotoxicity; however, the reasons for this are not yet fully understood. We report here a new signal transduction pathway involving protein kinase Cδ (PKCδ), NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS), that inhibits caspase-dependent cell death induced by TRAIL ligation in human malignant astrocytoma cells. In our experiments, TRAIL ligation-induced generation of intracellular ROS through caspase-dependent proteolytic activation of PKCδ and subsequent activation of the NOX4 complex. Suppression of intracellular ROS induction using various pharmacological inhibitors or PKCδ-or NOX4-specific RNA interference enhanced the enzymatic activity of caspase-3 by blocking the oxidative modification of its catalytic cysteine residue, resulting in marked augmentation of TRAIL-mediated cell death. These results collectively indicate that TRAIL-induced activation of PKCδ and NOX4 can modulate TRAIL-mediated apoptosis by promoting oxidative modification of active caspase-3 in a negative-feedback manner.