Abstract Body: Angiopoietin-like 4 (ANGPTL4) is a multifunctional glycoprotein that regulates several crucial biological processes, including glucose and lipid metabolism, vascular permeability, wound healing, angiogenesis, and tumorigenesis. ANGPTL4 has been linked to several diseases, including diabetes, retinal disorders, cancer progression, and metastasis, highlighting its significance in human health and disease. Diabetic macular edema (DME) is a primary cause of vision impairment in working-age Americans, with many patients experiencing insufficient response to current treatments targeting VEGFA. In the present study, we show that ANGPTL4, a HIF-1-regulated gene product, is overexpressed in the eyes of diabetic mice and patients with DME. Notably, ANGPTL4 and VEGF synergistically disrupt the retinal vascular barrier. Although ANGPTL4 slightly enhances VEGF receptor 2 tyrosine phosphorylation, its promotion of vascular permeability occurs independently of this receptor. Instead, ANGPTL4 binds directly to neuropilin 1 (NRP1) and NRP2 on endothelial cells, triggering rapid activation of the RhoA/ROCK signaling pathway and breakdown of endothelial cell junctions. A soluble fragment of NRP1 (sNRP1) blocked ANGPTL4-induced RhoA activation and endothelial cell permeability in vitro, as well as retinal vascular leakage in diabetic animals in vivo. Additionally, sNRP1 reduced the stimulation of endothelial cell permeability by aqueous fluid from patients with DME. These findings identify the ANGPTL4/NRP/RhoA pathway as a promising therapeutic target for treating DME.