Atherosclerosis (AS) is initiated by vascular endothelial cell injury, which is induced by lipid and protein oxidation. Oleoylethanolamide (OEA), a dietary fat-derived lipid, has shown atheroprotective effect. In vitro studies demonstrated that OEA showed cytoprotective effects on H2O2-induced primary cultured human umbilical vein endothelial cell (HUVEC) injury model. Further investigation of the cytoprotective effects of OEA demonstrated that OEA exerted its function by scavenging for reactive oxygen species, as well as increasing anti-oxidative enzymes, reducing lipid peroxidation, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and apoptosis-related proteins expression. The in vivo study using an ApoE-/- mouse model fed with high-fat diet for 8 weeks showed that OEA (10 mg/kg/day, i.g.) administration reduced blood lipid levels, prevented endothelial cell damage and inhibited early AS plaque formation. In conclusion, our results suggested that OEA exerted a pharmacological effect on ameliorating atherosclerotic plaque formation through the inhibition of oxidative stress-induced endothelial cell injury and therefore OEA can be a potential candidate drug for anti-atherosclerosis.