〔Abstract〕 Objective To investigate the differences in gut microbiota and metabolites between urban and rural middle school students and explore their significance in gut homeostasis, so as to establish a healthy lifestyle and diet for children. Methods Fecal samples were collected from middle school students in Hefei(n=14) and Jixi county(n=18, Southern Anhui), aged 13.0-13.5 years. Stool samples were sequenced by 16S ribosomal DNA identification(16S rDNA) and detected for non-targeted metabolites by liquid chromatograph mass spectrometer(LC-MS), followed by bioinformatic analysis. Results LachnoclostridiumandAnaerostipeswere dominant in the urban students that had been reported to be associated with colorectal cancer, atherosclerosis, depression and other disorders. In the village children,Ruminococcaceae UCG-002,BarnesiellaandEubacteriumdominated. An increased proportion of these microbes were related to metabolism of bile acids, short-chain fatty acids, lipid and carbohydrate decomposition, and play an important role in maintaining immune balance and physiological function. Additionally, significant differences in gut metabolites of the two groups were noted, mainly in arachidonic acid metabolism, platelet activation, serotonin metabolism, vitamin absorption, primary bile acid metabolism and other pathways. Conclusion Adolescent students of urban and mountainous areas differ in gut microbiota and metabolites. Rural children have a healthy bacterial flora and metabolites in guts due to a reasonable lifestyle and diet in comparison with the city children.