Vitreous fibrovascular membranes (FVMs), the hallmark of proliferative diabetic retinopathy (PDR), cause retinal hemorrhage, detachment, and eventually blindness. However, little is known about the pathophysiology of FVM. In this study, we used single-cell RNA sequencing on surgically harvested PDR-FVMs and generated a comprehensive cell atlas of FVM. Eight cellular compositions were identified, with microglia as the major cell population. We identified a GPNMB⁺ subpopulation of microglia, which presented both profibrotic and fibrogenic properties. Pseudotime analysis further revealed the profibrotic microglia was uniquely differentiated from retina-resident microglia and expanded in the PDR setting. Ligand-receptor interactions between the profibrotic microglia and cytokines upregulated in PDR vitreous implicated the involvement of several pathways, including CCR5, IFNGR1, and CD44 signaling, in the microglial activation within the PDR microenvironment. Collectively, our description of the novel microglia phenotypes in PDR-FVM may offer new insight into the cellular and molecular mechanism underlying the pathogenesis of DR, as well as potential signaling pathways amenable to disease-specific intervention.