Background— Neovascularization is potentially important for the treatment of ischemic heart and limb disease. We reported that reactive oxygen species (ROS) derived from gp91 phox (Nox2)-containing NAD(P)H oxidase are involved in angiogenesis in mouse sponge models as well as in vascular endothelial growth factor (VEGF) signaling in cultured endothelial cells. The role of gp91 phox -derived ROS in neovascularization in response to tissue ischemia is unknown, however. Methods and Results— Here, we show that neovascularization in the ischemic hindlimb is significantly impaired in gp91 phox−/− mice as compared with wild-type (WT) mice as evaluated by laser Doppler flow, capillary density, and microsphere measurements. In WT mice, inflammatory cell infiltration in the ischemic hindlimb was maximal at 3 days, whereas capillary formation was prominent at 7 days when inflammatory cells were no longer detectable. Increased O 2 ·− production and gp91 phox expression were present at both time points. The dihydroethidium staining of ischemic tissues indicates that O 2 ·− is mainly produced from inflammatory cells at 3 days and from neovasculature at 7 days after operation. Relative to WT mice, ischemia-induced ROS production in gp91 phox−/− mice at both 3 and 7 days was diminished, whereas VEGF expression was enhanced and the inflammatory response was unchanged. Infusion of the antioxidant ebselen into WT mice also significantly blocked the increase in blood flow recovery and capillary density after ischemia. Conclusions— gp91 phox -derived ROS play an important role in mediating neovascularization in response to tissue ischemia. NAD(P)H oxidases and their products are potential therapeutic targets for regulating angiogenesis in vivo.