*Aims of the study*: 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:* Obese and diabetic (ob/ob) mice were chronically fed with prebiotic-enriched diet (Ob-Pre) or with a normal chow 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. *Main findings:* We found that prebiotics reduced fat-mass development, improved glucose tolerance, alleviated oxidative-stress and low-grade inflammation and reduced gut permeability. We showed that the gut microbiota regulate stem-cell differentiation and increase the number of enteroendocrine L-cells. The dendrogram and principal-coordinate analysis of each method revealed two clusters corresponding to the dietary conditions. 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. *Principal 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