<p>Cholestatic liver disease is characterized by highly accumulated bile acids and cholangiocyte proliferation, resulting in the development of fibrosis, cirrhosis, and ultimately liver failure necessitating liver transplantation. Calcium (Ca²⁺) signaling is commonly dysregulated in cholestasis and serves as an important regulator mediating cell proliferation. However, the role of Ca²⁺-mediated cholangiocyte proliferation and treatment strategies in bile duct ligation (BDL)-induced liver injury remains poorly understood. By integrating transcriptomic analysis with molecular biology techniques, we explored the mechanisms of liver injury across BDL animal models, primary cholangiocytes, and human intrahepatic biliary epithelial cholangiocytes. Here, we found that a natural ingredient, senkyunolide A (SenA), effectively alleviated cholestasis-induced Ca²⁺ release from ER by inhibiting RYR channel, thereby preventing FIP200-mediated ER autophagy in response to Ca²⁺ transients on the cytosolic ER surface. Increased cytosolic Ca²⁺ further triggered ER stress, cholangiocyte cycle progression, and ductular reaction (DR). Importantly, SenA reversed the above process through its binding to chloride Channel CLIC Like 1 (CLCC1) for ubiquitination, thereby inhibiting CLCC1 activity and ER Ca²⁺ release. si-CLCC1-loaded liposomes targeting cholangiocytes enhanced the anti-DR effects of SenA. Collectively, by controlling ER release of Ca²⁺ in cholangiocytes, SenA presents potential for the development of therapeutic strategies aimed at addressing cholestatic fibrosis.</p><p></p>