Update information. Cholesterol gallstone is a common disease, but the pathogenesis of gallstone remains a mystery. The pathogenetic factors of gallstone disease include a genetic background, cholesterol condensation and bile supersaturation, while the risk factors may include fatty liver, diabetes, obesity and microbiome dysbiosis. Dysbiosis refers to altered bacterial composition and abundance, which is found to be associated with the pathogenesis of variety of diseases. Mass spectrometry-based proteomics emerges as a powerful tool to identify protein expressions of human samples. A number of proteomic studies have been performed to analyze human protein contents of bile samples. All these studies focus on the analysis of human proteins, and no study to date has been performed to analyze the protein expression of microbiome in bile samples associated with gallstone disease. In current study, we performed a label-free metaproteomic analysis of gallbladder bile samples from cholesterol gallstone disease patients and normal individuals. Maxquant was used in the comprehensive database searching to infer protein groups with quantitation. We attempt to investigate the dysbiosis changes, potential virulence factor of microbiome and host immune responses which contribute to gallstone formation and cholelithiasis.