Conjugated linoleyl beta-sitosterol (CLS) was prepared from beta-sitosterol and conjugated linoleic acid (CLA) via lipase-catalyzed synthesis in n-hexane in the presence of molecular sieves. Its plasma cholesterol-lowering properties were also studied. The optimal biosynthesis conditions were as follows: beta-sitosterol concentration of 50 micromol/mL, the molar ratio of CLA to beta-sitosterol of 1:1, the lipase concentration of 20 mg/mL, and 4 A molecular sieve concentration of 60 mg/mL in n-hexane at 50 degrees C with vigorous shaking of 150 rpm for 72 h, and the highest yield of CLS reached 72.6%. The isolated CLS mixed with mice feed had good cholesterol-lowering properties. It decreased serum total cholesterol (TC), serum triacylglycerols (TAGs), serum low-density lipoprotein cholesterol (LDL-C), atherogenic index (AI), liver weight (LW), liver index (LI), liver TC, and TAGs of mice, which was shown that CLS could prevent the formation of atherosclerosis and moderate the fat pathologic changes of liver. However, the HDL-C was not decreased, which proved the advantage of CLS over the other cholesterol-lowering products.