Background and Aims Studies have indicated that gallstone disease (GSD) is related to the alterations of the bile duct microbiome. However, the bile duct microbiome changes caused by abnormal function of the sphincter of Oddi (SO) that serves as the barrier between the intestinal tract and biliary tract are not fully known. Therefore, this study investigated the differences in clinical features and bile tract microbiotas between GSD patients with concomitant SO dysfunction (SOD) and GSD patients with normal SO function.Methods Fourteen patients with cholecystolithiasis and choledocholithiasis undergoing an emergency operation from 2019 to 2021 in Qinghai Provincial People's Hospital were prospectively recruited. Of the patients, 7 cases presented with SOD (SOD group), and 7 cases had normal SO function (normal SO group) according to the value of the SO pressure determined during operation. All patients' bile specimens were obtained before and three months after the operation. The 16S rRNA genes were extracted from the collected samples, and amplicon sequencing was performed. Then, the amplicon sequencing data of the 16S rRNA genes were analyzed using QIIME 2 microbiome bioinformatics platform.Results The preoperative white blood count in patients of the SOD group was significantly higher than that of the normal SO group (P<0.05). At the same time, the parameters of other laboratory tests and the general conditions showed no significant difference between the two groups of patients (all P>0.05). Different bile duct microbiotas were observed in all patients. The microbial abundance was highest in the postoperative samples from the SOD group, followed by postoperative samples from the normal SO group, preoperative samples from the SOD group, and preoperative samples from the normal SO group, successively. The distributions of species of the microbiota of the four groups of bile samples from the two groups were different from each other, and the bacterial communities involved in GSD patients with SOD were predominantly of Escherichia, Clostridium, and Enterococcus versus GSD patients with normal SO function.Conclusion GSD patients with SOD have a higher inflammatory burden and significantly increased microbial abundance. They present increased species of bacteria associated with cholelithiasis compared with GSD patients with normal SO function, which may more likely cause biliary tract infection and recurrent stones.