The purpose of this study was to investigate the effects of the active ingredients of barley lees on the physiological indexes, intestinal flora, and liver transcriptome of mice fed a high‐fat diet. Twenty‐four male C57BL/6J mice were randomly divided into 4 groups and fed the experimental diets for 5 weeks. The results showed that the fat‐soluble components of distillers' grains significantly reduced body weight, abdominal fat, perirenal fat, blood glucose, low‐density lipoprotein cholesterol, triglycerides, and total cholesterol in the high‐fat diet‐fed mice (p < .05), significantly decreased alanine aminotransferase and malondialdehyde levels, and significantly increased total superoxide dismutase, catalase, reduced glutathione and glutathione peroxidase levels (p < .05). At the phylum level, lipid‐soluble components significantly increased the abundance of Bacteroidetes and decreased the Firmicutes/Bacteroidetes ratio. At the genus level, the relative abundances of Bacteroidetes and Clostridium were increased. Transcriptomic analysis showed that lipid‐soluble components of spent grains reduced the mRNA expression of ANGPTL8, CD36, PLTP, and SOAT1 and increased the mRNA expression of CYP7A1 and ABCA1 in the cholesterol metabolism pathway, promoted the transport of cholesterol, and inhibited the absorption of cholesterol, which can decrease cholesterol levels by speeding up the conversion of cholesterol into bile acids.