The novel synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9-dien-28-oate (CDDO-Me) induces apoptosis in human cancer cells, showing potential as a cancer therapeutic agent. We previously demonstrated that CDDO-Me induces a c-Jun N-terminal kinase (JNK)-mediated DR5 expression and apoptosis. This study revealed the mechanism by which CDDO-Me induces JNK activation and subsequent DR5 upregulation and apoptosis. To determine whether CDDO-Me activates JNK and induces DR5 expression and apoptosis via oxidative stress by inducing the generation of reactive oxygen species (ROS), we examined the effects of various antioxidants on JNK activation, DR5 upregulation, and apoptosis induction by CDDO-Me. Thiol antioxidants, including N-acetyl-L-cycteine (NAC), glutathione (GSH) and dithiothrietol (DTT), abrogated CDDO-Me-induced apoptosis. In contrast, nonthiol antioxidants, including butylated hydroxyanisole (BHA), Trolox, mannitol, and Mn(II) tetra(4-benoic acid) porphyrin chloride (MnTBAP), failed to do so, with the exception of vitamin C (Vit C). Accordingly, only thiol antioxidants blocked JNK activation induced by CDDO-Me. CDDO-Me reduced intracellular levels of GSH; this reduction was abrogated only by thiol antioxidants and Vit C. However, CDDO-Me did not promote ROS generation. These results suggest that depletion of intracellular GSH, but not ROS generation, contributes to CDDO-Me-induced JNK activation and apoptosis, at least in our systems. Furthermore, these thiol antioxidants abrogated CDDO-Me-induced DR5 expression, whereas the GSH-depleting agent diethylmaleate also upregulated DR5 expression at concentrations that deplete intracellular GSH, demonstrating that GSH depletion can cause DR5 upregulation. Collectively, we conclude that CDDO-Me activates the JNK pathway via depletion of intracellular GSH, leading to DR5 upregulation and induction of apoptosis.