Introduction: Occult pancreaticobiliary reflux (OPBR) causes chronic inflammation of the gallbladder and associated mucosal malignant changes, but its pathogenic mechanism remains unclear. Objective The aim of this study was to investigate the pathogenic mechanism in OPBR through untargeted metabolomic analysis of bile collected during surgery. Methods Patients undergoing surgery for gallstones between December 2020 and May 2021 were eligible for inclusion. There were 30 OPBR patients and 338 non-OPBR patients, and 30 non-OPBR patients were randomly selected as normal controls. Metabolomics analysis was performed based on bile samples from patients in the OPBR group. Results The principal component analysis by liquid chromatography-mass spectrometry revealed that 30 metabolites were significantly different between patients with OPBR compared to controls. Among these characteristic metabolites, palmitic acid and arachidonic acid are known to be involved in gallstone formation, methionine and lysophosphatidylcholine are known to be associated with carcinogenesis. Conclusions The present study suggested that an LC/MS-based metabolomic studies provide an efficient and promising method for identifying potential biomarkers of OPBR. The identified characteristic metabolites in the OPBR group were significantly different to the control group.