Abstract: This study aimed to screen a strain with high cholesterol-lowering effect from traditional fermented yak yogurt in western Sichuan province and explore its therapeutic effect on hypercholesterolemia. This study focused on 295 strains of lactic acid bacteria isolated from traditional fermented yak yogurt in western Sichuan, and the lactic acid bacteria with high cholesterol-lowering ability were screened by ferric ammonium sulfate method and o-phthalaldehyde method, and the in vitro probiotic characterization test and safety evaluation were carried out, and the cholesterol-lowering effect of the strains was evaluated by establishing a mouse model of hypercholesterolemia induced by high-fat diet. The screening revealed four strains exhibiting favorable cholesterol-reducing efficiency and efficacy isolated from 295 lactic acid bacteria. Among the 4 strains, strain 166 had the highest survival rate in simulated gastric fluid tolerance assay test, and the number of viable bacteria remained above 5.3×10⁸ CFU/mL after 3 h of incubation in the simulated intestinal fluid, and it possessed a good bile salt tolerance performance, with a hydrophobic capacity of up to 62.3%, auto-aggregation capacity of 18.65%, which was identified as Limosilactobacillus fermentum. In addition, strain 166 was evaluated negatively for harmful metabolites, no hemolysis phenomenon, no gelatin liquefaction, and showed some resistance to eight antibiotics, which showed good in vitro probiotic properties and safety. In vivo experiments in animals showed that Limosilactobacillus fermentum 166 can significantly reduce the serum total cholesterol, triglycerides, low-density lipoprotein cholesterol levels and atherosclerosis index of mice, effectively increase the content of serum high-density lipoprotein cholesterol, and effectively improve the Lee’s index, liver index, Perirenal fat index and abdominal fat index. Therefore, Limosilactobacillus fermentum 166 has the potential to be developed as a functional probiotic product, providing data support for the prevention and adjunctive therapy of hypercholesterolemia.