Abstract: Objective To determine if the types of gallstones are associated with different bacterial profiles in bile.Methods Gallstones and bile samples were obtained from 50 patients undergoing elective cholecystectomy.Gallstone chemical composition was determined by infrared spectrometry analysis using KBr tablets.The bacteria composition of corresponding bile samples were analyzed by terminal restriction fragment length polymorphism (T-RFLP) genetic sequencing of 16s ribosomal DNA (rDNA) .Results Three types of gallstones were detected and the patients were grouped accordingly: pure cholesterol stones (n=25 patients) , pigment gallstones (n=11) , and mixed stones (n=14) .The overall positive detection rate of bacterial 16S rDNA gene fragments was 86% (43/50) , and the positive rate was not significantly different among the three groups.The microbiome of the pure cholesterol group was mainly composed of Klebsiella pneumoniae and Staphylococcus aureus.The bile pigment stones group was mainly composed of Proteus vulgaris.The mixed group was mainly composed of anaerobic digestion bacteria propionic acid bacteria, and Microbacterium flavum.Conclusion Different types of gallstones were associated with different bile microbiome profiles.