Growing evidence has indicated that supplementation with probiotics improves lipid metabolism. We aimed to investigate the beneficial effects of a probiotics mixture (PM) of three strains belonging to the species Bifidobacterium (B. longum, B. lactis, and B. breve) and two strains belonging to the species Lactobacillus (L. reuteri and L. plantarum) on cholesterol-lowering efficacy in hypercholesterolemic rats. A hypercholesterolemic rat model was established by feeding a high-cholesterol diet for eight weeks. To test the effects of PM on hypercholesterolemia, hypercholesterolemic rats were assigned to four groups, which were treated daily with low (1.65 × 10⁹ cfu/kg), medium (5.5 × 10⁹ cfu/kg), or high (1.65 × 10¹⁰ cfu/kg) doses of probiotic mixture or simvastatin for eight weeks. Significant reductions of serum total cholesterol (TC), triacylglycerol (TG), and low-density lipoprotein (LDL)-cholesterol levels, but increases of high-density lipoprotein (HDL)-cholesterol were observed after supplementation of PM in hypercholesterolemic rats. In PM-supplemented hypercholesterolemic rats, hepatic tissue contents of TC and TG also significantly decreased. Notably, the histological evaluation of liver tissues demonstrated that PM dramatically decreased lipid accumulation. For their underlying mechanisms, we demonstrated that PM reduced expressions of cholesterol synthesis-related proteins such as sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) in the liver. Taken together, these findings suggest that PM has beneficial effects against hypercholesterolemia. Accordingly, our PM might be utilized as a novel therapeutic agent for the management of hypercholesterolemia.