Cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) synthesizes NADPH, which is an essential cofactor for the generation of reduced glutathione (GSH), the most abundant and important antioxidant in mammalian cells. We investigated the role of IDPc in kidney ischemia/reperfusion (I/R) in mice. The activity and expression of IDPc were highest in the cortex, modest in the outer medulla and lowest in the inner medulla. NADPH levels were greatest in the cortex. IDPc expression in the S1-2 segments of proximal tubules was higher than in S3 which is much more susceptible to I/R. IDPc protein was also highly expressed in the mitochondria-rich intercalated cells of the collecting duct. IDPc activity was 10- to 30-fold higher than the activity of glucose-6-phosphate dehydrogenase, another producer of cytosolic NADPH, in various kidney regions. This study identifies that IDPc may be the primary source of NADPH in the kidney. I/R significantly reduced IDPc expression and activity and NADPH production, and increased the ratio of oxidized glutathione to total glutathione (GSSG/(GSH+GSSG)), resulting in kidney dysfunction, tubular cell damage and lipid peroxidation. In LLC-PK1 cells, up-regulation of IDPc by IDPc-gene transfer protected the cells against hydrogen peroxide, enhancing NADPH production, inhibiting the increase of GSSG/(GSH+GSSG), and reducing lipid peroxidation. IDPc down-regulation by siRNA treatment presented contrasting results to the up-regulation. In conclusion, these results demonstrate that IDPc is expressed differentially along tubules in patterns that may contribute to differences in susceptibility to injury, is a major enzyme in cytosolic NADPH generation in kidney, and is down-regulated with I/R.