Central to cellular responses to hypoxic environment is the hypoxia-inducible factor (HIF) transcriptional control system. A role for HIF-2alpha was investigated in a model of renal ischemia-reperfusion injury (IRI) associated with oxidative stress using HIF-2alpha knockdown mice. In these mice, HIF-2alpha expression was approximately one half that of wild-type mice, whereas HIF-1alpha expression was equivalent. HIF-2alpha knockdown mice were more susceptible to renal IRI, as indicated by elevated blood urea nitrogen levels and semiquantitative histologic analysis. Immunostaining with markers of oxidative stress showed enhanced oxidative stress in the kidney of HIF-2alpha knockdown mice, which was associated with peritubular capillary loss. Real-time quantitative PCR analysis showed decreased expression of antioxidative stress genes in the HIF-2alpha knockdown kidneys. Studies that used small interference RNA confirmed regulation of the antioxidative stress genes in cultured endothelial cells. Although HIF-2alpha knockdown mice were anemic, serum erythropoietin levels were not significantly increased, reflecting inappropriate response to anemia as a result of HIF-2alpha knockdown. Experiments that used hemodiluted mice with renal ischemia demonstrated that anemia of this degree did not affect susceptibility to ischemia. Knockdown of HIF-2alpha in inflammatory cells by bone marrow transplantation experiments demonstrated that HIF-2alpha in inflammatory cells did not contribute to susceptibility to renal IRI. Restoration of HIF-2alpha in endothelium by intercrossing with Tie1-Cre mice ameliorated renal injury by IRI, demonstrating a specific role of endothelial HIF-2alpha. These results suggest that HIF-2alpha in the endothelium has a protective role against ischemia of the kidney via amelioration of oxidative stress.