Diabetic retinopathy (DR), a leading cause of vision loss in working-age adults, induces mosaic patterns of vasculopathy that may be associated with spatial heterogeneity of intraretinal endothelial cells. We recently reported that secretogranin III (Scg3), a neuron-derived angiogenic and vascular leakage factor, selectively binds retinal vessels of diabetic but not healthy mice. Here, we investigated endothelial heterogeneity of three retinal vascular plexuses in DR pathogenesis and the therapeutic implications. Our unique in vivo ligand binding assay detected a 22.7-fold increase in Scg3 binding to retinal vessels of diabetic mice relative to healthy mice. Functional immunohistochemistry revealed that Scg3 predominantly binds to the DR-stressed CD31−deep retinal vascular plexus but not to the relatively healthy CD31+superficial and intermediate plexuses within the same diabetic retina. In contrast, VEGF bound to healthy and diabetic retinal vessels indiscriminately with low activity. FITC-dextran assays indicated that selectively increased retinal vascular leakage coincides with Scg3 binding in diabetic mice that was independent of VEGF, whereas VEGF-induced leakage did not distinguish between diabetic and healthy mice. Dose–response curves showed that the anti-Scg3 humanized antibody (hAb) and anti-VEGF aflibercept alleviated DR leakage with equivalent efficacies, and that the combination acted synergistically. These findings suggest: (i) the deep plexus is highly sensitive to DR; (ii) Scg3 binding to the DR deep plexus coincides with the loss of CD31 and compromised endothelial junctions; (iii) anti-Scg3 hAb alleviates vascular leakage by selectively targeting the DR-stressed deep plexus within the same diabetic retina; (iv) combined anti-Scg3 and anti-VEGF treatments synergistically ameliorate DR through distinct mechanisms.