The intestinal microbiota is important for tolerance induction through mucosal immunological responses. The composition of the gut microbiota of an infant is affected by environmental factors such as diet, disease and antibiotic treatment. However, already in utero, these environmental factors can affect the immunological development of the foetus and influence the future gut microbiota of the infant. To investigate the effects of antibiotic treatment of pregnant mothers on the offspring's gut microbiome and diabetes development, we treated non-obese diabetic (NOD) mice with a cocktail of antibiotics during gestation and the composition of the gut microbiota, diabetes incidence and major gut-related T lymphocyte populations were investigated in the offspring. We observed a persistent reduction in the general diversity of the gut microbiota in the offspring from NOD mothers treated with antibiotics during gestation compared with offspring from control mothers. In addition, by clustering the present bacterial taxa with principal component analysis, we found a differential clustering of gut microbiota in the offspring from NOD mothers treated with antibiotics during gestation compared with offspring from control mothers. Offspring from NOD mothers treated with antibiotics during gestation also showed some immunological alterations in the gut immune system, which could be related to the diversity of the gut microbiome and influence modulation of diabetes development at 20 weeks. Our data point out maternal derangement of the intestinal microbiota as a potential environmental risk factor for T1D development.