Retinopathy of prematurity (ROP) is a proliferative retinal vascular disorder that critically affects the visual development of premature infants, potentially leading to irreversible vision loss or even blindness. Despite its significance, the underlying mechanisms of this disease remain insufficiently understood. In this study, we utilized the oxygen-induced retinopathy (OIR) mouse model and conducted endothelial functional assays to explore the role of Sterol Regulatory Element-Binding Protein 1 (SREBF1) in ROP pathogenesis. SREBF1 expression levels, along with its downstream targets, were investigated through Western blotting, RT-qPCR, and immunofluorescence staining techniques. Furthermore, Co-Immunoprecipitation (Co-IP) was employed to examine the molecular mechanisms involved. Our results demonstrated a significant increase in SREBF1 expression in both the OIR mouse model and hypoxic primary human retinal microvascular endothelial cells (HRMECs). Interventions conducted both in vivo and in vitro showed notable efficacy in reducing pathological neovascularization. Importantly, we discovered that SREBF1 plays a key role in modulating lipid metabolism in HRMECs by regulating the expression of ACC1 and FASN, leading to cellular reprogramming. This reprogramming influences HRMEC proliferation, migration, and tube formation through the HIF-1α/TGF-β signaling pathway, ultimately contributing to pathological retinal neovascularization. These findings provide new insights into the role of SREBF1 in angiogenesis within the context of ROP, offering potential therapeutic targets for the management and treatment of this disease.Copyright © 2025. Published by Elsevier Ltd.