Removal of gluten from the diet can attenuate the intensity of autoimmunity and reduces the incidence of diabetes in the nonobese diabetic mouse. In this study, we tested whether a gluten-free diet could reduce autoimmunity in human preclinical type 1 diabetes. A trial consisting of 6 months of a gluten-free diet followed by another 6 months of normal gluten-containing diet was performed in 17 first-degree relatives with at least 2 antibodies among islet cell antibodies, glutamic acid decarboxylase autoantibodies, protein tyrosine islet antigen-2 autoantibodies, and insulin autoantibodies. Treatment effect was measured as autoantibody titers and acute insulin response to iv glucose tolerance test. Two subjects dropped out for lack of compliance to diet restrictions. Of the remaining 15 subjects, 3 developed diabetes. Autoantibody titers did not show significant changes after 6 months of gluten-free diet and again after return to normal diet. Acute insulin response to iv glucose tolerance test significantly increased in 12 of 14 subjects after the first 6 months of gluten deprivation (P = 0.04) and decreased in 10 of 13 subjects during the following 6-month period of normal diet (P = 0.07). Insulin sensitivity (homeostasis model assessment-insulin resistance) nonsignificantly improved after the gluten-free diet and subsequently decreased (P < 0.005) after 6 months of normal diet. These findings indicate that 6 months of gluten deprivation do not influence humoral autoimmunity, but may have a beneficial effect on preservation of beta-cell function in subjects at risk for type 1 diabetes.