Hypercholesterolemia is a significant risk factor for cardiovascular disease and metabolic disorders. Probiotics are the essential constituents of the gastrointestinal microbiota that provide health-promoting effects. Cholesterol-lowering activity is a specific property of probiotics, improving the cholesterol metabolism without adverse effects. Thus, the purpose of this study was to investigate the hypocholesterolemic effect of single and mixed cholesterol-lowering probiotic strains (includingLimosilactobacillus reuteriTF-7,Enterococcus faeciumTF-18, andBifidobacterium animalisTA-1) in high-cholesterol diet (HCD)-induced hypercholesterolemic rats. The results showed that the administration of single probiotics contributed to a reduction in the body weight gain, visceral organ indexes, hyperlipidemia, and hepatic steatosis and also an improvement in the gastrointestinal microbiota. Besides the effect of single cholesterol-lowering probiotics, three probiotics strains could also synergize their hypocholesterolemic effect when administered simultaneously. These findings indicate that three cholesterol-lowering probiotic strains are suitable for development as probiotic supplements to reduce the risk of diseases caused by cholesterol and exert health benefits with synergistic effect when administered simultaneously.