To investigate emerging clinical data suggesting a triglyceride (TAG)-lowering response to plant sterol (PS) therapy, we characterized changes in TAG metabolism in 16 C57BL/6J mice fed a basal control diet (CON) or the CON diet supplemented with 2% PS for 6 wk. PS consumption reduced (p<0.05) plasma (-28%) and hepatic (-30%) TAG concentrations compared with CON mice. PS consumption increased (p<0.05) hepatic lipogenic gene expression (sterol-regulatory-element-binding protein 1c, 2.4-fold of CON; fatty acid synthase, 6.5-fold of CON) and de novo lipogenesis (4.51+/-0.72 versus 2.82+/-0.61%/day) compared with CON. PS consumption increased (p<0.05) fecal palmitate and stearate excretion and reduced body weight gain compared with CON mice. Although no change in the transcription of intestinal fatty acid absorptive genes was observed, peroxisome proliferator-activated receptor alpha mRNA was reduced (p<0.05, 2.0-fold of CON) in the PS-fed mice. In conclusion, PS-fed C57BL/6J mice showed pronounced reductions in plasma and hepatic TAG concentrations despite increases in hepatic lipogenic gene expression and de novo lipogenesis. Interference with intestinal fatty acid/TAG metabolism as suggested by increased fecal fatty acid loss and reduced weight gain may be associated with the TAG-lowering response to PS consumption.