Cancer cells are usually under higher oxidative stress compared with normal cells. We hypothesize that introducing additional reactive oxygen species (ROS) insults or suppressing antioxidant capacity may selectively enhance cancer cell killing by oxidative stress-generating agents through stress overload or stress sensitization, whereas normal cells may be able to maintain redox homeostasis under exogenous ROS by adaptive response. Here, we show that parthenolide, a sesquiterpene lactone, selectively exhibits a radiosensitization effect on prostate cancer PC3 cells but not on normal prostate epithelial PrEC cells. Parthenolide causes oxidative stress in PC3 cells but not in PrEC cells, as determined by the oxidation of the ROS-sensitive probe H(2)DCFDA and intracellular reduced thiol and disulfide levels. In PC3 but not PrEC cells, parthenolide activates NADPH oxidase, leading to a decrease in the level of reduced thioredoxin, activation of phosphoinositide 3-kinase/Akt, and consequent FOXO3a phosphorylation, which results in the downregulation of FOXO3a targets antioxidant enzyme manganese superoxide dismutase and catalase. Importantly, when combined with radiation, parthenolide further increases ROS levels in PC3 cells whereas it decreases radiation-induced oxidative stress in PrEC cells, possibly by increasing reduced glutathione levels. Together, the results show that parthenolide selectively activates NADPH oxidase and mediates intense oxidative stress in prostate cancer cells by both increasing ROS generation and decreasing antioxidant defense capacity. The results support the concept of exploiting the intrinsic differences in the redox status of cancer cells and normal cells as targets for selective cancer killing.