RESEARCH PURPOSE: This investigation explored the therapeutic effects and mechanisms of Skullcapflavone II in hepatic fibrosis (HF). MATERIALS AND METHODS: The optimal concentration of Skullcapflavone II for LX2 hepatic stellate cells was determined using the CCK8 assay. EdU staining and flow cytometry were utilised to assess cell proliferation and G2/M phase arrest. Mice with carbon tetrachloride-triggered HF were administered Skullcapflavone II at low (15 mg/day), medium (30 mg/day), and high (60 mg/day) doses. Subsequently, hepatic damage and fibrosis were assessed via body weight, liver index, biochemical markers, and histopathological staining. Immunohistochemistry for Collagen I and α-SMA were utilised to examine hepatic stellate cell (HSC) activation. RNA sequencing was utilised to ascertain differentially expressed genes. Molecular docking simulated interactions among Skullcapflavone II and target proteins as well as outcomes were validated by implementing western blotting, immunohistochemistry, and RT-PCR. RESULTS: Skullcapflavone II inhibited LX2 cell proliferation and triggered G2/M phase arrest. Its optimal intervention concentration was 160 μM. In vivo, it ameliorated hepatic function, diminished serum indicators of fibrosis, and suppressed HSC activation. Diminished collagen sediment was validated utilising histopathological examination, whereas immunohistochemistry indicated decreased expression of Collagen I and α-SMA. Additionally, molecular docking showed strong binding of Skullcapflavone II to DNA replication-related proteins. Western blotting and RT-PCR implied that Skullcapflavone II disrupted DNA replication, which triggered G2/M arrest and hindered HSCs activation and proliferation. CONCLUSION: The abovementioned mechanisms of action of Skullcapflavone II substantiate its prospective clinical application against HF.