Recently, hepatic sinusoidal obstruction syndrome (HSOS) caused by herbal preparations containing pyrrolizidine alkaloids (PAs), such as Gynura Rhizoma (Tusanqi), has gained global attention. However, the lack of a reliable and reproducible animal model has greatly hampered mechanistic studies. Therefore, we aimed to establish a reproducible HSOS mouse model and investigate the hepatotoxic mechanism. The model was established by intragastrical administration of Gynura Rhizoma extract, i.e., 1.0 g extract/kg per day (equal to 16.7 g crude drug/kg per day based on extraction rate and 49.1 mg PA/kg per day based on the total PA content in the extract determined) for 40 successive days. Then, the mice were sacrificed, and their blood samples and livers were collected for analyses. Using hematoxylin-eosin (HE) and Masson staining, scanning electron microscopy imaging, clinical biomarkers, and other assays, we showed that the HSOS was successfully induced in our mouse model. Furthermore, we detected the key factors involved in liver fibrosis in the mice, revealing significantly increased hydroxyproline concentration; elevated expression of α-smooth muscle actin (α-SMA) and fibrosis-related genes such as Collagen-1, Collagen-3, Mmp2, Mmp13, Timp1, Timp3, and Activin, upregulated Smad3 phosphorylation, and increased serum TGF-β levels. Moreover, pro-inflammatory cytokines, including Tnf-α, Il-1β, and Il-6, were also increased in the model. All these results demonstrate the key roles of the TGF-β-Smad3 and inflammatory signaling pathways in this Gynura Rhizoma-induced HSOS mouse model, suggesting that blockade of fibrosis and/or inflammation should be an effective treatment for HSOS.