Plant sterols and stanols are structurally similar to cholesterol and when added to the diet they are able to reduce serum total- and LDL-cholesterol concentrations. They also lower serum triglyceride concentrations in humans, particularly under conditions of hypertriglyceridemia. The aim of this study was to unravel the mechanism by which plant sterols and stanols reduce serum triglyceride concentrations in high-fat diet (HFD) fed mice. Male C57BL/6J mice were fed HFD for 4 weeks. Subsequently, they received HFD, HFD supplemented with 3.1% plant sterol ester (PSE) or HFD supplemented with 3.1% plant stanol ester (PSA) for another three weeks. Both PSE and PSA feeding resulted in decreased plasma triglyceride concentrations compared with HFD, while plasma cholesterol levels were unchanged. Interestingly, hepatic cholesterol levels were decreased in the PSE/PSA groups compared with HFD and no differences were found in hepatic triglyceride levels between groups. To investigate the mechanism underlying the hypotriglyceridemic effects from PSE/PSA feeding, we measured chylomicron and VLDL secretion. PSE and PSA feeding resulted in reduced VLDL secretion, while no differences were found between groups in chylomicron secretion. In conclusion, our data indicate that plasma triglyceride-lowering resulting from PSE and PSA feeding is associated with decreased hepatic VLDL secretion.