Inflammatory diseases such as type 2 diabetes (T2D) in humans and mice are under the influence of the composition of the gut microbiota (GM). It was previously demonstrated that treating Lepob mice with antibiotics improved glucose tolerance. However, wild type C57BL/6J mice may also exhibit plasma glucose intolerance reminiscent of human T2D. We hypothesized that antibiotic treatment in C57BL/6 mice would have an impact on glucose tolerance without affecting weight and gut immunology. When compared to mice treated with erythromycin or the controls, treatment for five weeks with ampicillin improved glucose tolerance without significantly affecting the weight or the number of gut mucosal regulatory T cells, tolerogenic dendritic cells or T helper cells type 1. 16S rRNA gene based denaturing gradient gel electrophoresis profiles clearly clustered according to treatment and showed that antibiotic treatment reduced GM diversity. It is concluded that antibiotic treatment changes glucose metabolism as well as the composition of the GM in C57BL/6 mice, and that this does not seem to be correlated to weight development in the mice.