ARTICLE ABSTRACTPancreatic ductal adenocarcinoma (PDAC) frequently develops therapeutic resistances, which can be divided into extrinsic and intrinsic resistance. The extrinsic resistance that arises from the surrounding dense tumor stroma is much better understood. However, the mechanisms of intrinsic resistance are not well understood. Here, we report that reactive oxygen species (ROS) induced by gemcitabine treatment, a newly discovered cytotoxic activity, served as a probe in our study to reveal the mechanisms of the intrinsic therapeutic resistance. Our results showed that gemcitabine-induced ROS is generated by NOX and through the increase of p22−phox expression via NF-κB activation. As a feedback mechanism, nuclear translocation of Nrf2 stimulated the transcription of cytoprotective antioxidant genes, especially genes encoding enzymes that catalyze glutathione (GSH) production to reduce elevated ROS as an intrinsic resistance countermeasure. RNAi-mediated depletion of Nrf2 or addition of β-phenylethyl isothiocyanate inhibited the ROS detoxification process by reducing GSH levels, which, in turn, increased the efficacy of gemcitabine in vitro and in vivo. Thus, our study suggests that a redox-mediated pathway contributes to the intrinsic resistance of PDAC to gemcitabine and provides a basis for developing strategies to preferentially kill PDAC cells through ROS-mediated mechanism. The combination of gemcitabine and PEITC has a selective cytotoxic effect against pancreatic cancer cells in vivo and could thus prove valuable as a cancer treatment. Mol Cancer Ther; 14(3); 788–98. ©2014 AACR.