Objective To establish a model to evaluate the therapeutic effect of Biejiajian Pills (BJJP) on bile duct ligation (BDL)-induced liver fibrosis in rats, as well as to investigate the molecular mechanisms by which Biejiajian Pills (BJJP) alleviate trimetlylamineoxide (TMAO)-aggravated liver fibrosis in bile duct ligation (BDL) rats. Methods Firstly, for establishing the therapeutic model of BJJP for BDL-induced hepatic fibrosis in rats, 30 rats were randomly divided into the Sham group, the BDL group, the BJJP-L group, the BJJP-M group, the BJJP-H group. The deposition of collagen fibers was analyzed by polarized light image of Sirius Red staining. Expression levels of α-smooth muscle actin (α-SMA) and type I collagen α1 chain (COL1A1) proteins of liver tissue in each group of rats were detected by Immunohistochemical staining (IHC) and Western Blot. Rat feces of the Sham group, the BDL group, the BJJP-H group were collected for 16S rRNA high-throughput microbial sequencing and non-targeted metabolomics analysis. TMA and TMAO levels in rat feces and serum of the Sham group, the BDL group, the BJJP-H group were determined respectively by non-targeted and targeted metabolomics technologies. Then, for evaluating the therapeutic effects of BJJP on TMAO-aggravated hepatic fibrosis and exploring its potential molecular mechanisms, another 20 SD rats were randomly divided into the Sham group, the BDL group, the TMAO group, the TMAO+BJJP group. Changes in histomorphology and collagen fibers of liver tissue in each group of rats were objected by H E, Masson and Sirius Red staining. The expression levels of α-smooth muscle actin (α-SMA), flavin-containing monooxygenase 3 (FMO3), phosphorylated (p-) protein kinase B (AKT), and phosphorylated (p-) phosphatidylinositol 3-kinase (PI3K) in each group were quantitatively analyzed by Western Blot. Results Compared with the Sham group, the expression of α-SMA and COL1A1 protein of the BDL group were increased in the liver tissue. Compared with the BDL group, the results of BJJP-L, BJJP-M and BJJP-H group were opposite. BJJP could improve the intestinal flora and metabolic disorders of BDL rats, reduce the content of trimethylamine (TMA) in feces, and reduce serum TMAO levels in rats (all P<0.05). Compared with the Sham group, the BDL group exhibited disorganized hepatic cord arrangement, evident hepatocyte swelling, and extensive collagen fiber deposition, along with up-regulated protein expression of α-SMA, FMO3, p-AKT, and p-PI3K (all P<0.05). Conclusions BJJP can improve hepatic fibrosis of BDL rats, TMAO can aggravate hepatic fibrosis of BDL rats. BJJP may alleviate the hepatic fibrosis process of BDL rats by regulating intestinal flora TMAO.