Background A gluten-free diet reduces the incidence of diabetes mellitus in non-obese diabetic (NOD) mice, but the mechanism is not known. The aim of this study was to examine the possible influence of the diet on the caecal bacterial flora, which may affect the intestinal physiology and mediate disease prevention. Methods Two groups of NOD mice from the age of 3 weeks were fed either a gluten-free diet or a standard diet. Each diabetic mouse, when diagnosed, along with a non-diabetic mouse from the same diet group and two nondiabetic mice from the alternate diet group were euthanized and sampled for classical bacteriological examination. Results Nine out of 19 (47%) standard-fed mice and 1 out of 19 (5%) gluten-free-fed mice developed diabetes (p < 0.01). Mice on the gluten-free diet had significantly fewer aerobically (p < 0.01) and microaerophilically (p < 0.001) cultivated bacteria in their intestines than standard-fed mice. Non-diabetic mice also had significantly fewer microa erophilic and anaerobic bacteria than diabetic mice (p < 0.05). These differences were primarily due to a difference in the Gram-positive flora. Conclusions The gluten-free diet compared to the standard diet both qualitatively and quantitatively substantially altered the composition of the caecal bacterial flora in NOD mice. Although Gram-positive bacteria might influence the beta cells through certain digestive products, it is more likely to assume that any effect on diabetes incidence is immunological. Copyright (c) 2005 John Wiley & Sons, Ltd.