Objective To establish a model to evaluate the therapeutic effect of Biejiajian pills (BJJP) on bile duct ligation (BDL)-induced liver fibrosis in rats, and to investigate the molecular mechanisms by which BJJP alleviates trimethlylamine oxide (TMAO)-aggravated liver fibrosis in BDL rats. Methods A rat model of therapeutic BJJP for BDL-induced hepatic fibrosis was established. Thirty rats were divided randomly into Sham, BDL, and BJJP low-dose (-L), medium-dose (-M), and high-dose (-H) groups. Collagen-fiber deposition was analyzed by Masson and Sirius Red staining. Expression levels of α-smooth muscle actin ( α-SMA) and type Ⅰ collagen α1 chain (COL1A1) proteins in liver tissue in each group were detected by immunohistochemical staining and Western blot.Feces were collected from rats in the Sham, BDL, the BJJP-H groups for 16S rRNA high-throughput microbial sequencing and non-targeted metabolomics analysis. Serum and feces levels of trimethylamine ( TMA) and TMAO were determined in the Sham, BDL, and BJJP-H groups, respectively, by non-targeted and targeted metabolomics technologies. To investigate the therapeutic effects of BJJP on TMAO-aggravated hepatic fibrosis and its potential molecular mechanisms, a further 20 Sprague Dawley rats were divided randomly into Sham, BDL, BDL+TMAO, and BDL+ TMAO + BJJP-H groups. Changes in histomorphology and collagen fibers in liver tissue were detected by hematoxylin-eosin, Masson, and Sirius Red staining. Expression levels of α-SMA, flavin-containing monooxygenase 3(FMO3), phosphorylated (p-) protein kinase B (AKT), and p-phosphatidylinositol 3-kinase (PI3K) in each group were analyzed quantitatively by Western blot. Results Compared with the Sham group, liver levels of collagen fiber,α-SMA and COL1A1 protein were increased in the BDL group(P<0. 000 1, P<0. 000 1, P<0. 001). Compared with the BDL group, levels of collagen fiber in the BJJP-L, BJJP-M, and BJJP-H groups were significantly decreased(P<0. 05, P<0. 05, P<0. 001), as well as the levels of α-SMA protein and COL1A1 protein ( all P<0. 05). BJJP improved the intestinal flora and metabolic disorders in BDL rats, and reduced feces levels of TMA and serum levels of TMAO (all P<0. 05). Compared with the Sham group, rats in the BDL group exhibited disorganized hepatic cord arrangement, evident hepatocyte swelling, and extensive collagen-fiber deposition, along with up-regulation of α-SMA, FMO3, p-AKT, and p-PI3K protein expression(all P<0. 05). The above indicators in liver tissues of rats were further increased in the BDL+TMAO group compared with the BDL group(P<0. 001, P<0. 05, P<0. 000 1, P<0. 05). Compared with the BDL + TMAO group, rats in the BDL + TMAO + BJJP-H group exhibited more orderly hepatocellular arrangement, reduced collagen-fiber deposition(P<0. 000 1), and decreased protein expression of α-SMA, FMO3, p-AKT, and p-PI3K (P<0. 001, P<0. 01, P<0. 001, P<0. 05). Conclusions BJJP can improve hepatic fibrosis while TMAO can aggravate hepatic fibrosis in BDL rats. BJJP may alleviate the hepatic fibrosis process in BDL rats by regulating intestinal flora TMAO.