Background: The mouse model of high-fat diet-induced cholesterol gallstone is widely used in researches of pathogenesis, prevention and treatment of gallstones. Aims: To investigate the characteristics and hepatic transcriptomics of the mouse model of high-fat diet-induced cholesterol gallstone. Methods: Twenty male C57BL/6J mice were randomly allocated into chow diet (control) group and lithogenic diet (LD) group. After 8 weeks, the occurrence of gallstone was observed; the serum lipids and gallbladder bile lipids were detected; and the differentially expressed hepatic genes between the two groups were identified with Illumina NovaSeq sequencing systems. The enrichment analysis was mapped in GO and KEGG pathway databases. Real-time PCR was used to detect the expressions of genes related to bile acid synthesis in the liver. Results: The cholesterol gallstone formation rate was 100% in LD group, whereas no gallstone was observed in control group. Hepatomegaly and steatosis were obvious in mice of LD group. The serum levels of total cholesterol and triacylglycerol, as well as the cholesterol content and cholesterol saturation index of the gallbladder bile in LD group were significantly higher than those in control group (P<0.05). A total of 1 330 differentially expressed genes were identified by high-throughput sequencing and bioinformatics analysis. KEGG analysis showed that the differentially expressed genes were mainly enriched in lipid metabolism, bile secretion, and insulin secretion pathways. GO analysis showed that fatty acid metabolic process-related pathways were significantly enriched. Both hepatic transcriptomics analysis and real-time PCR showed that the expression levels of genes related to bile acid synthesis, including CYP7A1, CYP8B1 and CYP7B1 decreased significantly in the liver of LD group as compared with those of control group (P<0.05). Conclusions: The metabolism of cholesterol, bile acids and fatty acids is significantly disordered in mice with cholesterol gallstone. Transcriptomics analysis can screen out the differentially expressed genes that play roles in the formation of cholesterol gallstone, so as to provide references for studies focusing on these topics.