A high-throughput screening method for the selection of lactic acid bacteria with high cholesterol-degrading ability was established using the 12C6+ beam irradiation mutagenesis technique. This study aimed to breed excellent strains with high cholesterol-degrading capability and explore the mechanisms of cholesterol degradation by these strains. The mutated strains JMR-01 (Lactobacillus reuteri) and SN-2 (Lactobacillus rhamnosus) after irradiation were primarily screened for high cholesterol-degrading strains by optimizing the KENJI culture medium with colony diameter as an indicator. The primary screened strains were further screened using ammonium ferric sulfate method. The mechanisms of cholesterol degradation by lactic acid bacteria were further investigated by determining the total cholesterol degradation rate, the cholesterol degradation rate through co-precipitation and absorption. The results showed that after primary and secondary screening, four mutant strains with high cholesterol-degrading ability (JMR-01-1, JMR-01-2, SN-2-1, SN-2-9) were selected. The cholesterol-degrading ability of strain JMR-01-1 was increased by 9.7% compared to the original strain JMR-01 (P<0.05), and the cholesterol-degrading ability of strain SN-2-1 was increased by 11.5% compared to the original strain SN-2. The total cholesterol degradation rate, co-precipitation, and absorption of cholesterol degradation rate of the four mutant strains showed no significant difference in the co-precipitation effect on cholesterol compared to the original strains, but the relative absorption rate was significantly increased. This study optimized the screening method for cholesterol-degrading strains and demonstrated through the exploration of different cholesterol degradation pathways by lactic acid bacteria that this screening method can select strains with high cholesterol absorption capacity.