In this study, mice lacking the gp91 phox gene were used to address the role of NADPH oxidase in hyperhomocysteinemia-induced podocyte injury. It was found that a folate-free diet increased plasma homocysteine levels, but failed to increase O 2 − production in the glomeruli from gp91 phox gene knockout (gp91 −/−) mice, compared with wild-type (gp91 +/+) mice. Proteinuria and glomerular damage index (GDI) were significantly lower, whereas the glomerular filtration rate (GFR) was higher in gp91 −/− than in gp91 +/+ mice when they were on the folate-free diet (urine albumin excretion, 21.23 ± 1.88 vs 32.86 ± 4.03 μg/24 h; GDI, 1.17 ± 0.18 vs 2.59 ± 0.49; and GFR, 53.01 ± 4.69 vs 40.98 ± 1.44 μl/min). Hyperhomocysteinemia-induced decrease in nephrin expression and increase in desmin expression in gp91 +/+ mice were not observed in gp91 −/− mice. Morphologically, foot process effacement and podocyte loss due to hyperhomocysteinemia were significantly attenuated in gp91 −/− mice. In in vitro studies of podocytes, homocysteine was found to increase gp91 phox expression and O 2 − generation, which was substantially inhibited by gp91 phox siRNA. Functionally, homocysteine-induced decrease in vascular endothelial growth factor-A production was abolished by gp91 phox siRNA or diphenyleneiodonium, a NADPH oxidase inhibitor. These results suggest that the functional integrity of NADPH oxidase is essential for hyperhomocysteinemia-induced podocyte injury and glomerulosclerosis.