Abundant evidence indicates that lysophosphatidylcholine (LPC) is pro-inflammatory and atherogenic. In the vascular endothelium, LPC increases permeability as well as expression of proinflammatory molecules such as adhesion molecules and cytokines. Yet, mechanisms by which LPC mediate these activities remain unclear and controversial. Recent evidence implicates involvement of a novel subfamily of G protein-coupled receptors (GPR4, G2A, OGR1, and TDAG8) which are sensitive to lysolipids as well as protons. We previously reported that one of these receptors, GPR4, is selectively expressed by a variety of endothelial cells, and therefore, hypothesize that the LPC-stimulated endothelial barrier dysfunction is mediated through GPR4. We developed a peptide antibody against GPR4, which detected GPR4 expression in transfected COS 7 cells and endogenous GPR4 expression in endothelial cells by Western blot. Endothelial cells infected with a retrovirus containing small interference RNAi to GPR4 resulted in 40-50% decreased GPR4 expression, which corresponded with partial prevention of the LPC-induced a) decrease in transendothelial resistance, b) stress fiber formation and c) activation of RhoA. Further, co-expression of the siRNA-GPR4 with a siRNA-resistant mutant GPR4 fully restored the LPC-induced resistance decrease. However, extracellular pH of <7.4 did not alter baseline nor LPC-stimulated resistances. The results provide strong evidence that the LPC-mediated endothelial barrier dysfunction is regulated by endogenous GPR4 in endothelial cells, and suggest that GPR4 may play a critical role in the inflammatory responses activated by LPC.