The antiatherogenic properties of a novel dietary formula (PS-CO) of plant sterol esters of fatty acids, produced by enzymatic interesterification of plant sterols with canola oil (CO), in a CO matrix containing 1,3-diacylglycerol, were evaluated in apolipoprotein E-deficient mice. PS-CO consumption strongly tended to lower total plasma cholesterol levels by 21%, compared to the placebo group. Blood triglycerides were reduced by 38% and 36% compared to CO and placebo-fed mice, respectively. Serum lipid peroxide levels were lowered following PS-CO administration by 62% and 63%, compared to CO and placebo administration, respectively. Unlike CO supplementation, PS-CO consumption preserved serum paraoxonase (PON1) activity. Mouse peritoneal macrophages from PS-CO-fed mice exhibited reduced cellular uptake of oxidized-LDL compared to those from placebo-fed mice and demonstrated a tendency toward a decreased capability to release superoxide anions. These findings indicate that PS-CO supplementation is beneficial in reducing serum lipid levels, and serum and macrophage oxidative stress, thus contributing to the reduction in atherogenic risk factors.