Oxidized phospholipids may appear in the pulmonary circulation as a result of acute lung injury or inflammation. We have previously described barrier protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on human pulmonary endothelial cells (EC) mediated by small GTPases Rac and Cdc42. This work examined OxPAPC-induced focal adhesion (FA) and adherens junction (AJ) remodeling and potential interactions between FA and AJ protein complexes involved in OxPAPC-induced EC barrier enhancement. Immunofluorescence analysis, subcellular fractionation, and co-immunoprecipitation assays have shown that OxPAPC induced translocation and peripheral accumulation of FA complexes containing paxillin, focal adhesion kinase, vinculin, GIT1 and GIT2, increased association of AJ proteins VE-cadherin, p120-catenin, -, -, and -catenins, and dramatically enhanced cell junction areas covered by AJ. Co-immunoprecipitation, pulldown assays, and confocal microscopy studies have demonstrated that OxPAPC promoted novel interactions between FA and AJ complexes via paxillin and -catenin association, which was critically dependent upon Rac and Cdc42 activities and was abolished by pharmacological or siRNA-mediated inhibition of Rac and Cdc42. Depletion of -catenin using siRNA approach attenuated OxPAPC-induced paxillin translocation to the cell periphery, but also significantly decreased interaction of paxillin with AJ protein complex. In turn, paxillin knockdown by specific siRNA attenuated adherens junction enhancement in response to OxPAPC. These results show for the first time the novel interactions between FA and AJ protein complexes critical for EC barrier regulation by OxPAPC.