In this study, the biliary tolerance, acid resistance and cholesterol-lowering ability of 11 strains of Lactobacillus were studied, and the mechanism of removing cholesterol from Lactobacillus strains was explored. The cholesterol assimilation results determined by the difference in cholesterol content in the medium indicated that all Lactobacillus strains could assimilate cholesterol at a level of 12.03 to 32.25 μg/mL, and the cholesterol removal ability was related to the growth of the culture. The binding of cholesterol to lactobacilli cells was determined by growth, heat inactivation and dormant cells in phosphate buffer. The dry weight of cholesterol eliminated by heat and dormant cells was 0.79 to 3.82 mg/g; the dry weight of cholesterol cleared by growing cells was 4.53 to 16.03 mg/g. These results indicated that L. casei ASCC 292 and L. acidophilus 4962 strain exhibited good acid and biliary resistance, with the removal of most cholesterol from the medium. Lactobacillus strains could remove cholesterol by various mechanisms, and could be used as a dietary supplement to lower serum cholesterol in the body.