Dietary gluten, vitamin D3, and fish-oil are suggested to influence the incidence of autoimmune diabetes. To determine whether modification of their intake could reduce diabetes incidence and autoimmunity in mice, pups from female non-obese diabetic (NOD) mice were fed diets modified for protein source, fatty acid content, and/or vitamin D3 content and were followed for diabetes development, insulin autoantibodies (IAA), and insulitis. Replacement of wheat and barley with poultry as the major protein source significantly affected diabetes development. Diabetes onset was delayed and diabetes incidence was significantly reduced in female mice that received the wheat and barley protein-free diet throughout life (45% by age 32 weeks vs. 88% in control mice; P < 0.01), from weaning (42%; P < 0.005), or from 3 to 10 weeks of age only (36%; P < 0.01), and diabetes development was not completely restored by gliadin supplementation of the wheat and barley protein-free diet (58%; P < 0.05). Insulin autoantibodies (P < 0.01) and insulitis scores (P < 0.02) were reduced, and intra-pancreatic IL-4 mRNA increased (P < 0.05) in wheat and barley protein-deprived mice. Diabetes incidence was neither reduced by fish-oil or vitamin D3 supplementation alone, nor in mice fed a wheat and barley protein-free diet that was supplemented with fish-oil and vitamin D3. These data support a link between dietary wheat and barley proteins and the development of autoimmune diabetes.