Abstract: The activation of pancreatic stellate cells (PSCs) and the secretion of inflammatory factors play critical roles in the development of pancreatic fibrosis. While gambogic acid (GA), a flavonoid with anti-tumor properties, has been studied, its role in this process remains unclear. This study demonstrated that GA promoted YAP1 degradation and reduced its nuclear localization, thereby inhibiting PSC activation and the progression of pancreatic fibrosis. GA inhibited PSC proliferation, decreased α-smooth muscle actin (α-SMA) expression, and reduced lipid droplets in LTC14 and primary mouse PSCs (mPSCs). Additionally, GA suppressed the expression of inflammatory factors nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), nuclear factor erythroid 2-related factor 2 (NRF2), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and nuclear factor κB (NF-κB) in PSCs and counteracted the transforming growth factor (TGF)-β-induced increase in these proteins. GA also reduced collagen Ι and tissue inhibitor of metalloproteinase-1 (TΙMP1) expression, thereby attenuating fibrosis. Mechanistically, GA decreased YAP1 expression and nuclear translocation and reversed TGF-β-induced YAP1 upregulation. YAP1 overexpression abrogated GA’s inhibitory effects on PSC activation and inflammation. Furthermore, GA activated the Hippo pathway, increased phosphorylated (p)-LATS1 and p-YAP levels, and promoted ubiquitin-mediated YAP1 degradation. In vivo studies confirmed that GA inhibited dibutyltin dichloride (DBTC)-induced pancreatic fibrosis via suppressing YAP1 and NF-κB in BALB/c mice. In conclusion, GA activates the Hippo pathway and promotes YAP1 translocation to the cytoplasm, leading to its degradation and subsequent inhibition of PSC activation and fibrosis. These findings highlight the critical role of ubiquitin-mediated YAP1 degradation in regulating PSC activity and offer novel insights into the therapeutic potential of GA for treating pancreatic fibrosis.