Pancreatic islet autoimmunity leading to type 1 diabetes could be triggered by viruses in genetically susceptible individuals. Rotavirus (RV), the most common cause of childhood gastroenteritis, contains peptide sequences highly similar to T-cell epitopes in the islet autoantigens GAD and tyrosine phosphatase IA-2 (IA-2), suggesting T-cells to RV could trigger islet autoimmunity by molecular mimicry. We therefore sought an association between RV infection and islet autoantibody markers in children at risk for diabetes who were followed from birth. There was a specific and highly significant association between RV seroconversion and increases in any of these antibodies: 86% of antibodies to IA-2, 62% to insulin, and 50% to GAD first appeared or increased with increases in RV IgG or IgA. RV infection may therefore trigger or exacerbate islet autoimmunity in genetically susceptible children.