〔Abstract〕 Objective To explore the effect of liver-specific knockout of transcription factor EB(Tfeb) on high-fat diet(HFD)-induced hepatic steatosis in mice. Methods Wild-type C57 BL/6 J mice and liver-specific Tfeb knockout C57 BL/6 J mice were fed with HFD or normal chow for 12 weeks, respectively, and then the serum triglyceride(TG), total cholesterol(TC), monocyte chemoattractant protein-1(MCP-1), tumor necrosis factor-α(TNF-α), aspartate aminotransferase(AST) and alanine transaminase(ALT) were measured in each group of mice; Western blot was used to detect Tfeb protein expression levels in liver tissues of mice in each group, HE staining was used to monitor histopathological changes in liver tissues of mice in each group, oil red O staining was used to monitor lipid deposition in liver tissues of mice in each group, and F4/80 fluorescence staining was used to monitor macrophage infiltration in liver tissues of mice in each group. Results There was no expression of Tfeb gene in liver of liver-specific knockout Tfeb mice, suggesting that the effect of Tfeb gene knockout in liver tissue was better. Compared with the normal control group, the expression of Tfeb protein in the liver tissue of the model control group was down-regulated.Compared with the normal control group, both the HE staining results and the oil red O staining results showed that the liver specific Tfeb caused lipid deposition and liver lobule disorder in mice, which was similar to the liver changes in the model control mice, however, liver-specific knockout of Tfeb mice at 12 weeks of HFD had more severe liver lipid deposition and hepatic lobular structural disorder. The results of F4/80 fluorescence staining indicated that the specific knockout of liver Tfeb could aggravate the infiltration of macrophages in the liver of mice induced by high-fat feeding. At the same time, the serological test results indicated that compared with the normal control group, the serum levels of TC, TG, TNF-α, MCP-1, AST and ALT in the liver-specific knockout Tfeb group and the model control group increased, and these changes were further elevated in Tfeb knockout mice after HFD feeding. Conclusion Liver-specific knockout of Tfeb aggravates HFD-induced hepatic steatosis in mice.