Polydatin is a stilbene that has been demonstrated to regulate lipid, cholesterol, and glucose metabolism in humans. However, its potential role in cholesterol gallstone formation remains uncertain. C57BL/6 mice were fed a lithogenic diet (LD) and administered polydatin via intragastric administration. At the end of the 8-week study period, the animals were euthanized in order to collect bile/serum samples and gallbladder/liver tissues for subsequent analysis. studies were conducted in which human intrahepatic biliary epithelial cells (HIBECs) were exposed to lipopolysaccharide (LPS) for a period of 24 h. Subsequently, the culture supernatant and cells were harvested for further analysis. The results demonstrated that polydatin markedly reduced cholesterol gallstone formation, attenuated pathological alterations in the gallbladder and liver tissues, and improved lipid profiles in serum and bile samples. Moreover, polydatin exhibited anti-inflammatory properties, regulated cholesterol metabolism-related genes, and activated the PPAR-γ signaling pathway in mice fed an LD diet. In HIBECs, polydatin treatment prevented LPS-induced inflammatory cytokine release, dysregulation of cholesterol metabolism-related genes, and inactivation of the PPAR-γ pathway. This study is the first to demonstrate that polydatin prevents cholesterol gallstone formation by regulating cholesterol metabolism via the PPAR-γ signaling pathway.