INTRODUCTION: Growing evidence demonstrates that the gut microbiota plays a critical role in the development of obesity, type-2 diabetes and insulin resistance. Prebiotics have been shown to improves these disorders by increasing gut peptides secretion (i.e., glucagon-like peptide-1 and 2 (GLP-1 and GLP-2)). However, the exact composition of bacterial communities and the specific mechanisms involved in the increased intestinal GLP-1 and GLP-2 content remain unknown. OBJECTIVES: To investigate deep and comprehensive analysis of gut microbial communities and biological parameters (glucose homeostasis, metabolic stresses, and gut permeability) following prebiotics administration in obese and diabetic mice. METHODS/DESIGN: Obese and diabetic ob/ob mice were chronically fed with prebiotic-enriched diet (Ob-Pre) or with a standard diet (Ob-CT). Extensive gut microbiota analyses coupled with metabolic parameters were performed. We combined multiple molecular methods, including quantitative-PCR, bar-coded pyrosequencing of the 16S-ribosomal-RNA (rRNA), and phylogenetic-microarrays. Mice were subjected to glucose tolerance and gut permeability tests. Tight-junctions localization and distribution as well as intestinal enteroendocrine L-cells were quantified by immunohistological analyses. RESULTS: We found that prebiotics reduced fat-mass development, improved glucose tolerance, alleviated oxidative-stress and low-grade inflammation and reduced gut permeability. We found that the gut microbiota regulate stem-cell differentiation and increase L-cells number. Analyses revealed two clusters corresponding to the dietary conditions. Prebiotics treatment significantly reduced Firmicutes/Bacteroidetes ratio. 102 distinct 16S-rRNA sequences were affected by the prebiotic treatment, 16 of which displayed a >10-fold change in abundance after the treatment. Importantly, multivariate-analyses revealed strong correlations between specific bacteria and the observed biological footprints. CONCLUSIONS: We conclude that specific gut microbiota modulation target enteroendocrine cells, improve gut barrier integrity, and glucose intolerance. By profiling the gut microbiota, we demonstrated that specific bacteria can modulate the phenotype of obese mice, and identified novel bacterial targets that may affect host-metabolism and enteroendocrine cells numbers in obesity and diabetes