Abstract: Gypensapogenin I (GI) is a new dammarane type triterpenoid saponin extracted from Gynostemma pentaphyllum. This study explored the therapeutic potential and mechanism of GI on liver fibrosis (LF). LF is a key pathological stage of chronic liver disease, and the current treatment is limited. In the mouse LF model induced by carbon tetrachloride (CCl₄) and the LX-2 hepatic stellate cell model activated by transforming growth factor-β1 (TGF-β1), GI showed significant antifibrotic effects in vitro and in vivo, and could reduce liver injury, inflammation, collagen deposition and cell activation. Mechanistic studies have shown that GI exerts its effect by directly targeting MST1 protein. The direct and specific interaction between GI and MST1 was confirmed by surface plasmon resonance (SPR), drug affinity reaction target stability(DARTS) and cell thermal transition analysis (CETSA). Site directed mutagenesis further identified Arg181 as a key residue necessary for the stable binding of GI to MST1. This binding activated the Hippo signaling pathway, promoted the phosphorylation of MST1, LATS1/2 and YAP, resulting in the retention and inactivation of YAP in the cytoplasm. More importantly, after knockdown of STK4 gene encoding Mst1 by siRNA, the antifibrotic effect of GI was completely abolished, thus establishing MST1 as its essential functional target. In summary, this study found that GI is a novel MST1 agonist, which alleviates LF by targeting Arg181 residue, restoring Hippo/YAP pathway homeostasis and inhibiting hepatic stellate cell activation, which makes GI a promising candidate drug in the treatment of LF.