OBJECTIVE Microbial factors influence the development of diabetes in NOD mice. Studies in germ-free animals have revealed important roles of microbiota in the regulation of Th17 and forkhead box P3 (FOXP3)(+) T regulatory (Treg) activation in the intestine. However, the effects of intestinal microbiota in immune regulation and diabetes development in NOD mice are still poorly understood. METHODS A colony of germ-free NOD mice was established to evaluate the effects of intestinal microbiota on regulatory immunity in the gut, and on the development of insulitis and diabetes in NOD mice. RESULTS Diabetes developed in roughly equal numbers in germ-free and specific pathogen-free NOD mice. Insulitis was accentuated in germ-free NOD mice; yet insulin preservation was unaltered. Germ-free NOD mice showed increased levels of Il17 (also known as Il17a) mRNA in the colon, and of Th17 and Th1 cells in the mesenteric and pancreatic lymph nodes, while Foxp3 mRNA and FOXP3(+) Tregs were reduced. In the islet infiltrates, FOXP3(+)CD4(+) T cells were slightly increased in germ-free mice. B cells appeared less activated in the peritoneum and were less abundant in islet infiltrates. CONCLUSIONS These results indicate that lack of intestinal microbiota promotes an imbalance between Th1, Th17 and Treg differentiation in the intestine. This imbalance is associated with accelerated insulitis, but intact recruitment of FOXP3(+) Tregs into islets, suggesting: (1) a microbial dependence of local induction of Treg in the gut and draining lymph nodes; but (2) a potentially compensatory function of naturally occurring Tregs in the islets, which may help control diabetogenic T cells.