Identifying effective anti-fibrotic therapies is a major clinical need that remains unmet. In the present study, roseotoxin B was shown to possess an improving effect on cholestatic liver fibrosis in bile duct-ligated mice, as proved by histochemical and immunohistochemical staining, hepatic biochemical parameters, and TUNEL apoptotic cell detection in tissue sections. Using cellular thermal shift assay, computational molecular docking, microscale thermophoresis technology, and surface plasmon resonance biosensor, we confirmed that PDGFR-β was a direct target of roseotoxin B in fibrotic livers. Of note, human tissue microarrays detected pathologically high expression of p-PDGFR-β in liver samples of ~80% of patients with liver fibrosis and cirrhosis. PDGF-B/PDGFR-β pathway promotes transdifferentiation and excessive proliferation of hepatic stellate cells (HSCs), which is a very crucial driver for liver fibrosis. Meaningfully, roseotoxin B blocked the formation of PDGF-BB/PDGFR-ββ complex by targeting the D2 domain of PDGFR-β, thereby inhibiting the PDGF-B/PDGFR-β pathway in HSCs. In summary, our study provided roseotoxin B as a unique candidate agent for the treatment of liver fibrosis.