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This experiment was designed to evaluate the potential for dietary alfalfa saponins (AS) to influence cholesterol (Ch) concentrations and the mRNA expressions of the ATP-binding cassette (ABC) that concludes transporters G5 (ABCG5) and G8 (ABCG8) in the livers of laying hens. Hundred and fifty laying hens (27 weeks old) were divided into five treatment groups randomly. Five replicates were assigned to each treatment group consisting of six birds per replicate. The same basic diet was used for all groups with supplementation of 0 (control), 60, 120, 240, and 480 mg/kg of AS, which was given ad libitum to hens for 60 days. The results showed that AS in the diets of laying hens could effectively reduce egg Ch concentrations, and increase the excretion of bile acid by the liver with the expression levels of ABCG5 and ABCG8 improving. Our study found that dietary AS could effect cholesterol metabolism by up-regulating the expression of G5 and G8 in hens, and that AS is a potential agent for reducing Ch concentrations and 120 mg/kg of AS in the diet was suitable and effective for laying hens.