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Growing evidence indicates an important link between gut microbiota, obesity, and metabolic syndrome. Alterations in exocrine pancreatic function are also widely present in patients with diabetes and obesity. To examine this interaction, C57BL/6J mice were fed a chow diet, a high-fat diet (HFD), or an HFD plus oral vancomycin or metronidazole to modify the gut microbiome. HFD alone leads to a 40% increase in pancreas weight, decreased glucagon-like peptide 1 and peptide YY levels, and increased glucose-dependent insulinotropic peptide in the plasma. Quantitative proteomics identified 138 host proteins in fecal samples of these mice, of which 32 were significantly changed by the HFD. The most significant of these were the pancreatic enzymes. These changes in amylase and elastase were reversed by antibiotic treatment. These alterations could be reproduced by transferring gut microbiota from donor C57BL/6J mice to germ-free mice. By contrast, antibiotics had no effect on pancreatic size or exocrine function in C57BL/6J mice fed the chow diet. Further, 1 week vancomycin administration significantly increased amylase and elastase levels in obese men with prediabetes. Thus, the alterations in gut microbiota in obesity can alter pancreatic growth, exocrine function, and gut endocrine function and may contribute to the alterations observed in patients with obesity and diabetes. [ABSTRACT FROM AUTHOR]