Diabetic retinopathy (DR) is the leading cause of acquired blindness in middle-aged people. The complex pathology of DR is difficult to dissect, given the convoluted cytoarchitecture of the retina. Here, we performed single-cell RNA sequencing (scRNA-seq) of retina from type 2 diabetic model induced in leptin receptor-deficient (db/db) and control db/m mice with the aim of elucidating the factors mediating the pathogenesis of DR. We identified eleven cell types and determined cell type-specific expression of DR-associated loci via genome-wide association study-based enrichment analysis. DR also impacted cell type-specific genes and altered cell-cell communication. Based on the scRNA-seq results, retinaldehyde-binding protein 1 (RLBP1) was investigated as a promising therapeutic target for DR. Retinal RLBP1 expression was decreased in diabetes, and its overexpression in Müller glia mitigated DR-associated neurovascular degeneration. These data provide a detailed analysis of the retina under diabetic and normal conditions, revealing new insights into pathogenic factors that may be targeted to treat DR and related dysfunctions.© 2021 by the American Diabetes Association.