Abnormal bile acid metabolism is associated with various diseases, including acute pancreatitis (AP), although its beneficial effects have not been fully elucidated. This study aimed to investigate the relationship between deoxycholic acid (DCA) and AP. We designed a comprehensive analysis pipeline involving two-sample Mendelian randomization (MR) and targeted bile acid metabolomics based on a bi-center population to identify potential metabolic influencers of AP. These analyses were complemented by in vivo and in vitro experimental studies to assess the effects of metabolites, followed by integrated analysis and prediction of target molecules to explore the potential mechanisms underlying the protective effects of DCA. In MR studies, we identified a causal relationship between DCA and AP. A population-based targeted bile acid metabolomics showed that DCA levels were significantly reduced during the acute phase of AP compared with those in healthy controls and were associated with acute respiratory distress syndrome and infectious pancreatic necrosis. In the cholecystokinin-induced pancreatic acinar cell injury model and the caerulein-induced AP model, DCA administration alleviated pancreatic acinar cell injury, improved the pathological manifestations of pancreatic tissues, and reduced serum lipase and amylase levels. Integrated analysis of pancreatic transcriptomic data and DCA target proteins prediction by PharmMapper revealed significant alterations in the PPAR signaling pathway. Furthermore, Western blot and immunofluorescence assays confirmed that DCA likely mediates its protective effects through this mechanism. For the first time, we found that DCA is closely associated with the development of AP using a public database and a dual-center clinical cohort and that supplementation with DCA can improve acinar cell necrosis in AP, representing a promising novel strategy for the prevention and treatment of AP.