Background <p>Lysophosphatidyl choline acyltransferase 3 (LPCAT3) is crucially involved in the remodeling of phospholipids in the membranes through incorporation of arachidonic acid (ARA; 20:4). The ARA-derived eicosanoids aggravate leukocyte adhesion, inflammation, vascular dysfunction, thrombosis, and atherogenesis. This study found that LPCAT3 modulates lipid rafts and contributes to the raft assembly/organization essential for cytokine signaling.</p> Methods <p>RNAi-dependent silencing of LPCAT3 in the endothelial cells. EPA and DHA enrichment in the cells. Lipid raft isolation and analysis of proinflammatory signaling molecules. Diet-induced atherosclerosis in the mice. LPCAT3 siRNA lipid nanoparticles/ EPA, DHA therapy.</p> Results <p>RNAi-dependent silencing of LPCAT3 inhibits TNFα-induced translocation &amp; ubiquitination of TNFR1-signaling complex into the lipid rafts. This is associated with the attenuated NF-κB activation, synthesis of cell-adhesion molecules, cytokines, leukocyte adhesion, vascular permeability and endothelial dysfunction. Intriguingly, LPCAT3 inhibition resulted in significantly greater accretion of EPA and DHA in the PC and PE at the expense of ARA, and potentially decreased the ARA-derived eicosanoids in the vascular endothelium. Therapeutic administration of LPCAT3 siRNA-lipid nanoparticles in the high fat fed- mice markedly lowered the plasma glucose, insulin, proinflammatory cytokines, eicosanoids, and attenuated the plaque formation in the aorta. Co-treatment of LPCAT3 siRNA-lipid nanoparticles with EPA/DHA significantly elevated the accretion of EPA/DHA levels in the heart tissues and nullified the plaque development in the mice.</p> Conclusions <p>Our data revealed that LPCAT3-dependent remodeling of lipid rafts is essential for the TNF-induced signal transduction, NF-kB activation, and vascular inflammation. Administration of LPCAT3 siRNA-lipid nanoparticles and EPA/DHA is an effective strategy to combat atherosclerosis.</p>