The purpose of this study was to examine the effect of yeast β-glucan, as compared to β-glucan from a botanical source, on blood lipids and intestinal probiotic bacteria in hypercholesterolemic male Wistar rats. The barley, isolated barley-derived β-glucan, and isolated yeast-derived β-glucan were used in cholesterol AIN-93 diets (~3 g β-glucan/100 g diet) of the rats. After an 8-week feeding trial, the barley, barley-derived β-glucan and yeast-derived β-glucan diets fed to rats caused significant (p<0.05) reductions in the levels of total cholesterol (11%, 15%, 9%, respectively) and LDL-cholesterol (24%, 33%, 20%, respectively) in serum as compared to the control group. Among the test diets, the barley-derived β-glucan diet had the greatest cholesterol-lowering effect. There was a significant increase in the HDL-cholesterol/total cholesterol ratio in the serum of all test diets-fed rats (p<0.05) and a significant decrease in the LDL-cholesterol/HDL-cholesterol ratio in the serum of barley and barley-derived β-glucan-fed rats (p<0.05), as compared to the control group. The number of Lactobacilli was significantly higher (p<0.05) in comparison to the control group for the barley and barley-derived β-glucan groups (11.8 and 12.6 log10 counts/g wet fresh caecum, respectively). The same trend was also found for Bifidobacteria (12.3 and 13.1 log10 counts/g wet fresh caecum, respectively). The botanical and yeast glucans worked in the same manner, leading to a conclusion that the cholesterol-lowering potency of β-glucan is not dependent on its source. On the other hand, the probiotic activity of β-glucan does depend on the source.