The intestinal transporter PEPT1 mediates the absorption of di- and tripeptides originating from breakdown of dietary proteins. Whereas mice lacking PEPT1 did not display any obvious changes in phenotype on a high-carbohydrate control diet (HCD), Pept1^−/− mice fed a high-fat diet (HFD) showed a markedly reduced weight gain and reduced body fat stores. They were additionally protected from hyperglycemia and hyperinsulinemia. Energy balance studies revealed that Pept1^−/− mice on HFD have a reduced caloric intake, no changes in energy expenditure, but increased energy content in feces. Cecal biomass in Pept1^−/− mice was as well increased twofold on both diets, suggesting a limited capacity in digesting and/or absorbing the dietary constituents in the small intestine. GC-MS-based metabolite profiling of cecal contents revealed high levels and a broad spectrum of sugars in PEPT1-deficient mice on HCD, whereas animals fed HFD were characterized by high levels of free fatty acids and absence of sugars. In search of the origin of the impaired digestion/absorption, we observed that Pept1^−/− mice lack the adaptation of the upper small intestinal mucosa to the trophic effects of the diet. Whereas wild-type mice on HFD adapt to diet with increased villus length and surface area, Pept1^−/− mice failed to show this response. In search for the origin of this, we recorded markedly reduced systemic IL-6 levels in all Pept1^−/− mice, suggesting that IL-6 could contribute to the lack of adaptation of the mucosal architecture to the diets.