The compartmentalization and transport of proteins and solutes across the endothelium is a critical biologic function altered during inflammation and disease leading to pathology inmultiple disorders. The impact of tissue damage and subsequent extracellularmatrix (ECM) fragmentation in regulating this process is unknown. We demonstrate that the collagen-derived matrikine acetylated proline-glycine-proline (N-a-PGP) serves as a critical regulator of endothelial permeability.N-α-PGP activates human endothelial cells via CXC-chemokine receptor 2 (CXCR2), triggeringmonolayer permeability through a discrete intracellular signaling pathway. In vivo, N-α-PGP induces local vascular leak after subcutaneous administration and pulmonary vascular permeability after systemic administration. Furthermore, neutralization of N-α-PGP attenuates lipopolysaccharide-induced lung leak. Finally, we demonstrate that plasma from patients with acute respiratory distress syndrome (ARDS) induces VE-cadherin phosphorylation in human endothelial cells, and this activation is attenuated by N-α-PGP blockade with a concomitant improvement in endothelial monolayer impedance. These results identify N-α-PGP as a novel ECM-derivedmatrikine regulating paracellular permeability during inflammatory disease and demonstrate the potential to target this ligand in various disorders characterized by excessive matrix turnover and vascular leak such as ARDS.