Proliferative retinopathies, such as those complicating prematurity and diabetes, are major causes of blindness. A prominent feature of these retinopathies is excessive neovascularization, which is orchestrated by the hypoxia-induced vascular endothelial growth factor (VEGF) stimulating endothelial cells, and the integrin-mediated adhesive interactions of endothelial cells with extracellular matrix components like fibronectin (FN). Recently, we demonstrated that α-defensins interfere with α5β1-FN interactions and dependent endothelial cell functions (FASEB J., 2004, 18:1306–8). Here, α-defensins were studied in hypoxia-induced proliferative retinopathy. In vitro, α-defensins specifically inhibited α5β1-integrin dependent migration of bovine retinal endothelial cells (BREC) to FN, attenuated the VEGF-stimulated increase in endothelial permeability, and blocked BREC proliferation and capillary sprout formation in three-dimensional fibrin-matrices. An upregulation of β1-integrin and FN was observed in the retinal vessels in the mouse model of hypoxia-induced retinal angiogenesis. Systemic and ocular administration of α-defensins reduced retinal neovascularization by 45% and 60%, respectively, and this effect was comparable to the inhibitory effect of α5β1-blocking antibody. α-defensins were detected in human diabetic retinas but were absent in retinas of eyes removed because of trauma. Together, these data show that α-defensins inhibit pathological retinal neovascularization in vivo and may provide a clinically efficient strategy against proliferative retinopathies.