EphrinB2 ligands and EphB4 receptors are expressed on endothelial cells (EC) of arteries and veins, respectively, and are essential for vascular development. To understand how these molecules regulate retinal neovascularization (NV), we evaluated their expression in a model of oxygen-induced retinopathy (OIR). EphrinB2 and EphB4 were expressed on arterial and venous trunks, respectively, and on a subset of deep capillary vessels. EphB4 expression was reduced following hyperoxia, while ephrinB2 expression remained unaltered. In addition, a subset of EphB4-positive veins regressed in a caspase-3-dependent manner during hyperoxia. Arteriovenous malformations were also observed with loss of arterial-venous boundaries. Finally, both ephrinB2 and EphB4 were expressed on a subset of neovascular tufts following hyperoxia. These data confirm the contribution of ECs from both venous and arterial origins to the development of retinal NV.