Overproduction of reactive oxygen species (ROS), through either endogenous or exogenous sources, could induce DNA damage, and accumulation of DNA damage might lead to multistep carcinogenesis. The antioxidative effects of vitamin D have been suggested by epidemiological and many in vitro and in vivo laboratory studies. While exploring the antioxidative effects of vitamin D in prostate cells, we found that the active form of vitamin D, 1 alpha, 25-dihydroxyvitamin D(3) (1,25-VD), can protect nonmalignant human prostate epithelial cell lines, BPH-1 and RWPE-1, but not malignant human prostate epithelial cells, CWR22R and DU 145, from oxidative stress-induced cell death. Glucose-6-phosphate dehydrogenase (G6PD), a key antioxidant enzyme, was dose- and time-dependently induced by 1,25-VD. Mechanistic studies using chromatin immunoprecipitation (ChIP) assay revealed that a direct repeat-3 (DR3) vitamin D response element located in the first intron of the G6PD genome can be bound by liganded vitamin D receptor, thereby regulating G6PD gene expression. Increasing G6PD activity and glutathione level by 1,25-VD can scavenge cellular ROS. Moreover, the protective effects of 1,25-VD were abolished by dehydroepiandrosterone, a noncompetitive inhibitor of G6PD activity. Together, our results showed that 1,25-VD can protect nonmalignant prostate cells from oxidative stress-induced cell death by elimination of ROS-induced cellular injuries through transcriptional activation of G6PD activity. The antioxidative effect of vitamin D strengthens its roles in cancer chemoprevention and adds to a growing list of beneficial effects of vitamin D against cancer. (c) 2008 Wiley-Liss, Inc.