Background <p>Proliferative diabetic retinopathy (PDR) is a leading cause of irreversible blindness worldwide, characterized by pathological neovascularization and progressive retinal fibrosis. Although anti-VEGF therapy effectively suppresses neovascularization, it does not address fibrovascular membranes (FVMs) formation and may paradoxically accelerate membrane contraction, increasing the risk of tractional retinal detachment. The lack of effective anti-fibrotic strategies highlights the importance to elucidate the key cellular mechanisms underlying retinal fibrosis in PDR.</p> Methods <p>We performed single-cell RNA sequencing (scRNA-seq) on primary retinal tissues from PDR organ donors and non-diabetic controls, generating a single-cell transcriptomic atlas of the human PDR retina. Critical findings were validated by immunofluorescence, in vivo and in vitro functional assays. Stromal-specific Sox15 conditional knockout mice in a two-stage laser-induced fibrosis model and siRNA-mediated knockdown in fibroblasts, and intravitreal administration of the EDNRB antagonist were performed.</p> Results <p>scRNA-seq of primary human PDR retinas generated the first comprehensive single-cell transcriptomic atlas of human PDR, revealing profound alterations in cellular composition including microglial expansion and Müller glia reprogramming as central neuroinflammatory features of PDR, and selective loss of metallothionein enriched rods subpopulation and S-cones. Notably, We identified a stromal cell population that was enriched in PDR retinas, localized in FVMs, and characterized by high SOX15 expression. Cell-cell communication analysis revealed that EDN1-EDNRB signaling between these stromal cells and Müller glia represented the most prominent intercellular interaction in PDR. Mechanistically, SOX15 regulates EDN1 transcription through binding to its promoter, driving EDN1 expression that activates EDNRB-expressing retinal macroglia to promote reactive gliosis and retinal fibrosis. Stromal-specific Sox15 conditional knockout significantly attenuated fibrotic lesion formation in vivo, and PDR-derived fibrocytes with elevated SOX15 exhibited enhanced fibroblast differentiation and EDN1 secretion, providing translational validation in human disease. Intravitreal EDNRB blockade with BQ-788 significantly reduced fibrotic area in a murine fibrosis model.</p> Conclusions <p>This study identifies key pathological features of the human PDR retina, including pervasive neuroinflammation and selective loss of photoreceptor subpopulations, and delineates the SOX15–EDN1-EDNRB axis as a novel mechanism driving retinal fibrosis. Targeting this axis may represent a promising anti-fibrotic strategy for advanced PDR.</p> Graphical Abstract <p></p>