Objective: The Notch signaling pathway is closely related to biliary fibrosis. Previous studies have shown that Astragaloside (AS) can prevent the progression of cholestatic liver fibrosis. The purpose of this study is to observe the effect of AS on the regulation of Notch signaling pathway in biliary fibrosis. Methods: Cholestatic liver fibrosis was established by common bile duct ligation (BDL) in rats. Two weeks after BDL, the rats were randomly divided into a model group (i.e., BDL), an Astragalosides group (AS), and a sorafenib (SORA) positive control group and treated for 3 weeks. Bile duct proliferation and liver fibrosis were determined by tissue staining. Protein and gene expression were determined by immunostaining, immunoblotting and RT-PCR, respectively. Activation of the Notch signaling pathway was evaluated by analyzing expressions of Notch-1, -2, -3, -4, Jagged (JAG)1, Delta like (DLL)-1, -3, -4, Hes1, Numb and RBP-Jκ. Statistical analysis of variance analysis, q test, P < 0.05 showed that the difference was statistically significant. Results: (1) AS significantly reduced the deposition of collagen and the Hyp content of liver tissue (500.15 ± 86.10 vs. 625.72 ± 105.62, P = 0.031), and inhibited the activation of hepatic stellate cells. (2) AS significantly decreased the protein and mRNA expressions of transforming growth factor (TGF)-β1 (1.02±0.15 vs. 1.89±0.36, P = 0.007; 1.17±0.18 vs. 1.68±0.29, P = 0.013, respectively) and α-smooth muscle actin (α-SMA, 0.41±0.11 vs. 0.72±0.16, P = 0.003; 1.71±0.57 vs. 2.68±0.46, P = 0.008, respectively) compared with BDL group. In contrast, AS significantly enhanced expression of the Smad 7 protein compared with the BDL group (0.72±0.008 vs. 0.33±0.001, P = 0.005). AS also reduced biliary epithelial cell proliferation. AS reduced the mRNA levels of CK7, CK8 and CK18 (1.31±0.39 vs. 2.63±0.82, P = 0.009; 0.71±0.09 vs. 0.87±0.08, P = 0.031; 2.56±0.32 vs. 3.41±0.39, P = 0.010, respectively) and reduced the positive areas of CK19 and OV6 (62 337.17±21 873.38 vs. 22 5472.67±26 933.63, P = 0.000; 92 237.43±15 894.11 vs. 171 298.13±61 761.37, P = 0.000, respectively). (3) The mRNA expression of Notch-2, -3, -4 and JAG1 were significantly reduced in the AS group compared to the BDL group (1.07±0.19 vs. 1.51±0.28, P = 0.044; 0.99±0.24 vs. 1.18±0.10, P = 0.043; 1.36±0.42 vs. 3.40±0.44, P = 0.048; 2.62±0.43 vs. 3.73±0.83, P = 0.046, respectively). In contrast, the mRNA level of Numb was clearly enhanced after AS treatment (0.90±0.05 vs. 0.75±0.11, P = 0.019). In addition, consistent with the mRNA levels, the protein expressions of Notch-2, -3, -4 and JAG1 were reduced significantly (1.27±0.18 vs. 1.71±0.26, P = 0.004; 0.99±0.11 vs. 4.38±0.60, P = 0.001; 1.76±0.32 vs. 4.01±0.74, P = 0.002; 1.62±0.33 vs. 2.74±0.63, P = 0.002) and the Numb protein level was increased significantly (1.50±0.15 vs. 0.85±0.11, P = 0.001) in AS group compared with BDL group. Conclusion: AS may prevent cholestatic liver fibrosis via inhibition of the Notch signaling pathway, thereby inhibiting the abnormal proliferation of biliary epithelial cells. Results indicate that AS may be a potential treatment for cholestatic liver disease.