The incidence of diabetic nephropathy has been increasing. Studies have shown that oxidative stress (due to increased oxidant production and/or decreased antioxidant activity) is a critical underlying mechanism. The principal intracellular reductant is NADPH whose production is mainly dependent on glucose-6-phosphate dehydrogenase (G6PD) activity. Our work in cultured cells previously showed that high glucose caused activation of protein kinase A (PKA) and subsequent phosphorylation and inhibition of G6PD activity and hence decreased NADPH. The purpose of this study was to determine whether these findings occur in diabetic rats (induced by streptozotocin) as compared to control. G6PD activity and accordingly NADPH levels and glutathione levels were significantly decreased in diabetic kidneys as compared to control. Lipid peroxidation was significantly increased which correlated with decreased G6PD activity (r=0.48). G6PD expression was significantly reduced which correlated with decreased G6PD activity (r=0.72). PKA activity and serine phosphorylation of G6PD were significantly increased and were closely correlated with decreased G6PD activity (r=0.51 for PKA activity; r=0.93 for serine phosphorylation of G6PD). Insulin treatment and/ or correction of hyperglycemia ameliorated the changes caused by diabetes. In conclusion, chronic hyperglycemia caused inhibition of G6PD activity via decreased expression and increased phosphorylation of G6PD, which therefore led to increased oxidative stress.