〔Abstract〕 ObjectiveTo investigate the role of transcription factor EB (TFEB) -autophagy pathway in rifampicin-induced liver injury and its possible mechanism. Methods Forty 6-8-week-old C57/BL6 mice were randomly divided into five groups: control group, model group, TFEB low-dose agonist group, TFEB high-dose agonist group, and autophagy agonist group, with 8 mice in each group. Except for the control group, the other four groups were given rifampicin 200 mg/（kg·d）by gavage daily. TFEB agonist was administered intraperitoneally at a low dose of 20 mg/kg and a high dose of 50 mg/kg for 7 days at 1 h after rifampicin administration. Autophagy agonist was administered by gavage at a dose of 10 mg/kg 6 h before rifampicin administration on day 1. The experiment was completed 7 days after modeling. The degree of liver injury was evaluated by detecting liver function indexes and liver pathological changes. Western blot was used to detect the protein expression total TFEB, chelator 1（p62）, microtubule-associated protein light chain 3（LC3）, benzyl chloride 1（Beclin-1）, sodium taurocholate co-transporting polypeptide（NTCP）and bile salt export pump（BSEP）levels in liver nucleus/liver tissue were quantified. Results Compared with the control group, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), and total bile acid (TBA) in the model group increased ( P < 0.05), and obvious pathological changes were observed in the liver. Compared with the model group, the high dose and low dose of TFEB agonist and autophagy agonist groups had reductions in the above indicators ( P < 0.05). Compared with the low-dose TFEB agonist group, the high-dose TFEB agonist group had reductions in the above indicators ( P < 0.05). The proportion of TFEB in the nucleus was (1.0±0.10) in the control group, (0.6±0.05) in the model group, (0.8±0.08) in the low-dose agonist group, and (0.9±0.07) in the high-dose agonist group ( P < 0.05). Autophagy agonist group (0.7±0.06) ( P < 0.05). Compared with the control group, the levels of NTCP and BSEP in the liver of the model group decreased ( P < 0.05), and the expression of NTCP and BSEP in the TFEB low-dose and high-dose agonist groups were restored, and the expression of NTCP and BSEP in the autophagy agonist group also increased ( P < 0.05). Compared with the control group, the protein expression levels of TFEB, LC3-Ⅱ/LC3-Ⅰand Beclin-1 in the liver tissue of the model group significantly decreased ( P < 0.05), while the protein expression level of p62 significantly increased ( P < 0.05). Compared with the model group, the protein expression levels of TFEB, LC3-Ⅱ/LC3-Ⅰand Beclin-1 in the liver tissue of the TFEB agonist high-dose group, low-dose group and autophagy agonist group increased ( P < 0.05), while the protein expression level of p62 decreased ( P < 0.05). Conclusion TFEB can improve rifampicin-induced liver injury by activating autophagy pathway, and the main mechanism may be related to the up-regulation of NTCP and BSEP expression.