Vasohibin is a recently identified protein that is up-regulated in cultured vascular endothelial cells by vascular endothelial growth factor and fibroblast growth factor 2. It inhibits endothelial cell migration, proliferation, and tube formation, and suppresses angiogenesis in chick chorioallantoic membrane, after subcutaneous implantation of matrigel, and in a tumor xenograft model. This has led to the hypothesis that vasohibin functions as a negative feedback inhibitor of angiogenesis. In this study, we tested that hypothesis in a well-characterized model of retinal neovascularization. In ischemic retina, increased expression of VEGF was accompanied by elevation of vasohibin mRNA and blocking of the increase in vegf mRNA with vegf siRNA significantly attenuated the rise in vasohibin mRNA. In transgenic mice in which the rhodopsin promoter drives expression of VEGF in the retina, there was also a significant increase in vasohibin mRNA. In mice with ischemic retinopathy, there was increased expression of vasohibin in vascular endothelial cells, and vasohibin knockdown caused an increase in neovascularization. Conversely, intraocular injection of recombinant vasohibin or an adenoviral vector containing a vasohibin expression cassette strongly suppressed retinal neovascularization in mice with ischemic retinopathy. Knockdown of vasohibin mRNA in ischemic retina had no significant effect on vegf or vegf receptor 1 mRNA levels but caused a significant elevation in the level of vegf receptor 2 mRNA. These data support the hypothesis that vasohibin acts as a negative feedback regulator of neovascularization in the retina and suggest that suppression of VEGF receptor 2 may play some role in mediating its activity.