BACKGROUND: Barley, which is rich in ␤-glucans, is known to exhibit hypocholesterolemic effect. OBJECTIVE: The study aimed to investigate the hypocholesterolemic effect of yogurt containing barley ␤-glucan (BBG) and probiotic bacteria in rats fed on a cholesterol-enriched diet. METHODS: The methodology was based on adding of 0.75% BBG to skim milk (SM) powder. Four treatments of yogurt were formulated, wherein the first treatment was produced from SM without the addition of BBG and fermented by yogurts starter (YS). The second treatment was produced from SM with the addition of 0.75% BBG, and fermented by YS. The third treatment was produced from SM without the addition of 0.75% BBG, and fermented by Bifidobacterium lactis plus Lactobacillus acidophilus. The fourth treatment was produced from SM with addition of 0.75% BBG, and fermented by Bifidobacterium lactis plus Lactobacillus acidophilus. All formulations were evaluated for their effect on plasma lipids, liver lipids, lipid peroxidation, and the fecal excretion of bile acids in rats. RESULTS: The results indicated that yogurt containing probiotic bacteria and BBG was more effective in lowering of plasma and liver cholesterol levels than other treatments. The fecal excretions of bile acids and lipid peroxidation were markedly promoted in yogurt formulated with BBG and probiotic bacteria compared with the positive control group. The results showed an inverse relationship between the fecal excretions of bile acids and the levels of total cholesterol in the plasma from rats fed on a high-cholesterol diet.