Background It remains controversial whether avoidance of dietary diabetogenic triggers, such as cow's milk proteins, can prevent type 1 diabetes in genetically susceptible individuals. Here, different extensive casein hydrolysates (HC) and single amino acid (AA) formulations were tested for their effect on mechanisms underlying autoimmune diabetes pathogenesis in diabetes-prone BioBreeding rats. Intestinal integrity, gut microbiota composition and mucosal immune reactivity were studies to assess whether these formulations have differential effects in autoimmune diabetes prevention. Methods Diabetes-prone BioBreeding rats received diets in which the protein fraction was exchanged for the different hydrolysates or AA compositions, starting from weaning until the end of the experiment (d150). Diabetes development was monitored, and faecal and ileal samples were collected. Gut microbiota composition and cytokine/tight junction mRNA expression were measured by quantitative polymerase chain reaction. Cytokine levels of ileum explant cultures were measured by ELISA, and intestinal permeability was measured in vivo by lactulose-mannitol assay. Results Both HC-diet fed groups revealed remarkable reduction of diabetes incidence with the most pronounced effect in Nutramigen (R)-fed animals. Interestingly, AA-fed rats only showed delayed autoimmune diabetes development. Furthermore, both HC-fed groups had improved intestinal barrier function when compared with control chow or AA-fed animals. Interestingly, higher IL-10 levels were measured in ileum tissue explants from Nutramigen (R)-fed rats. Beneficial gut microbiota changes (increased Lactobacilli and reduced Bacteroides spp. levels) were found associated especially with HC-diet interventions. Conclusions Casein hydrolysates were found superior to AA-mix in autoimmune diabetes prevention. This suggests the presence of specific peptides that beneficially affect mechanisms that may play a critical role in autoimmune diabetes pathogenesis. Copyright (c) 2012 John Wiley & Sons, Ltd.