Vascular integrity and the maintenance of blood vessel continuity are fundamental features of the circulatory system maintained through endothelial cell-cell junctions. Defects in endothelial barrier become an initiating factor in several pathologies including ischemia/reperfusion, tumor angiogenesis, pulmonary edema, sepsis and acute lung injury. Better understanding of mechanisms stimulating endothelial barrier enhancement may provide novel therapeutic strategies. We have previously reported that oxidized phospholipids (OxPAPC) promote endothelial cell (EC) barrier enhancement both in vitro and in vivo. This study examined the initiating mechanistic events triggered by OxPAPC to increase vascular integrity. Our data demonstrate that OxPAPC directly binds the cell membrane localized chaperone protein, GRP78, associated with its cofactor, HTJ-1. OxPAPC binding to plasma membrane localized GRP78 leads to GRP78 trafficking to caveolin-enriched microdomains (CEM) on the cell surface and consequent activation of sphingosine 1-phosphate receptor 1 (S1P-R1), Src and Fyn tyrosine kinases and Rac1 GTPase, processes essential for cytoskeletal reorganization and EC barrier enhancement. Using animal models of acute lung injury with vascular hyper-permeability, we observed that HTJ-1 knockdown blocked OxPAPC protection from IL-6 and ventilator-induced lung injury. Our data indicate for the first time an essential role of GRP78 and HTJ-1 in OxPAPC-mediated CEM dynamics and enhancement of vascular integrity.