Indirect evidence is accumulating for an association between antibiotic use, especially in early childhood, and long-term immunologic health. The authors evaluated the association between antibiotic use in childhood and subsequent development of type 1 diabetes. A nationwide cohort study of all Danish singleton children born during 1995-2003 (n = 606,420) was conducted. Incidence rate ratios for type 1 diabetes comparing children according to antibiotic use were estimated. Antibiotic use was classified according to class, number of uses, and age at use. Use of any antibiotic was not associated with type 1 diabetes (rate ratio = 1.16, 95% confidence interval: 0.91, 1.50). Evaluation of type 1 diabetes risk according to number of courses of any antibiotic yielded no association between antibiotic use and type 1 diabetes, with an increase in rate ratio per course of 1.02 (95% confidence interval: 0.97, 1.07). No specific class of antibiotics was associated with type 1 diabetes, no specific age of use was associated with type 1 diabetes, and no specific age at onset of type 1 diabetes was associated with antibiotics. In a large nationwide prospective study, no association between antibiotic use and type 1 diabetes was found among Danish children.