Hyperglycemia impacts retinal vascular function and promotes the development and progression of diabetic retinopathy, which ultimately results in growth of new blood vessels and loss of vision. How high glucose affects retinal endothelial cell (EC) properties requires further investigation. Here we determined the impact of high glucose on mouse retinal EC function in vitro. High glucose significantly enhanced the migration of retinal EC without impacting their proliferation, apoptosis, adhesion, and capillary morphogenesis. The enhanced migration of retinal EC under high glucose was reversed in the presence of the antioxidant N-acetylcysteine suggesting increased oxidative stress under high glucose conditions. Retinal EC under high glucose conditions also expressed increased levels of fibronectin, osteopontin, and v3 integrin and reduced levels of thrombospondin-1. These changes were concomitant with sustained activation of the downstream prosurvival and promigratory signaling pathways including Src kinase, PI3K/Akt1/eNOS, and ERKs. The sustained activation of these signaling pathways was essential for enhanced migration of retinal EC under high glucose conditions. Together our results indicate the exposure of retinal EC to high glucose promotes a promigratory phenotype. Thus, alterations in the proangiogenic properties of retinal EC during diabetes may contribute to the development and pathogenesis of diabetic retinopathy.