High cholesterol level in serum is a major factor of influence for coronary heart disease. The cholesterol-lowering ability of lactic acid bacteria (LAB) without side effects makes them more and more attractive. Seventy-nine strains of LAB isolated from fermented food were screened in vitro for their ability to assimilate cholesterol. Then, ten strains which exhibited higher ability of cholesterol assimilation were investigated with the characteristics of acidic resistance, bile salt tolerance, and cell adhesion. According to the results, the best strain LP96 was picked out, and used to evaluate its effects on the high-cholesterol diet-fed rats. The results demonstrated that the levels of serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol, and liver TC and TG were reduced significantly in the groups that received the strain LP96 solution compared with the model group, and that the serum high-density lipoprotein cholesterol levels were increased without any significant difference. Furthermore, LP96 also showed good antioxidative activity and improvement of intestinal microbial balance in the rats. Thus, LP96 may be a promising probiotics with potential cholesterol-lowering ability.