In obesity and diabetes, abnormal gut microbiota and increased gut permeability (leaky gut) are common risk factors which in turn contributes in increasing chronic low grade inflammation. However, precise mechanism(s) whether and how gut microbiota contributes in induction of leaky gut remain largely unknown. Here we, demonstrated that obese/ diabetic gut microbiota not only associated with leaky gut and inflammation, but it plays a causative role to induce leaky gut and inflammation. Using fecal microbiota transplantation, intestinal organoids and human epithelial Caco2 cell systems, we demonstrated that obese microbiota dramatically reduces intestinal barriers such as tight junction proteins (TJPs), that are associated with increased leaky gut and inflammation. Intestinal epithelial TJPs are important structural proteins that holds epithelial cells tight enough to regulate non‐specific permeability of microbial/ food and other antigens from intestinal lumen, thereby, keep a check on inflammatory reactions in the mucosal immune system. We also demonstrated that gut microbiota metabolites that are enriched in obese/ diabetic gut contributes in reducing the expression of TJPs and increasing leaky gut. To decipher the mechanism, we found that obese/diabetic microbiota derived metabolites increases an array of miRNAs (including miR‐10a and miR101a‐3p) that downregulates TJPs expression, which in turn results leaky gut and chronic inflammation in diabetes and obesity. Results demonstrating that obese/diabetic microbiota and its metabolites are causative to increase leaky gut and inflammation by activating miRNAs that disrupts TJPs, are not only of great significance to understand the underlying biology of leaky gut and inflammation in obesity and diabetes, but will also pave the ways to develop novel strategies to prevent/ treat these debilitating public health diseases.Support or Funding InformationWe thank funding from the Department of Defense, National Institutes of Health and Wake Forest School of Medicine, and wonderful support from other team members.