AbstractPurpose Proangiogenic factors, VEGF and Cyr61, drive aberrant retinal neovascularization in diabetic retinopathy (DR). Their expression is regulated by the transcription factor Sp1. Several layers of the retina participate in the secretion of these factors; however, the contribution of the retinal pigment epithelium (RPE) to the pathogenesis of diabetic retinopathy has remained largely overlooked. Our objective was to characterize the Sp1‐dependent production of VEGF and Cyr61 in models of the diabetic retina, specifically focusing on the RPE, with the ultimate goal of identifying potential novel therapeutic targets for the treatment of DR.Methods Glucose‐treated ARPE‐19 (human retinal pigment epithelial cells) and TR‐iBRB (rat retinal microendothelial cells) were assayed for levels of VEGF and Cyr61 by qPCR, Western blot, and tubule formation assays. RNAi was used to deplete cells of Sp1. Binding of Sp1 to VEGF and Cyr61 promoters was monitored by chromatin immunoprecipitation (ChIP). Immunohistochemistry for VEGF and Cyr61 was done in diabetic mouse retinas.Results Glucose treatment caused increased VEGF and Cyr61 transcript and protein, and Sp1 depletion abrogated these changes in both cell types. ChIP analysis showed glucose‐induced increase in the Sp1 binding to VEGF and Cyr61 promoters. Additionally, expression of both factors was increased in the RPE of diabetic mice.Conclusion VEGF and Cyr61 are upregulated in ARPE‐19 and TR‐iBRB in hyperglycemia, which coincides with elevated Sp1 binding at their promoters. Depletion of Sp1 significantly reduced their aberrant expression. Sp1 may participate in the pathogenesis of diabetic retinopathy via upregulation of these proangiogenic genes in the RPE as well as in the vascular retina.