Breast-feeding has been suggested to have a protective effect against the development of type 1 diabetes. In the present study, we investigated the relation between duration of breast-feeding and beta-cell autoantibodies in 5-year-old non-diabetic children who participated in a prospective population-based follow-up study (the All Babies in Southeast Sweden study). Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA) and the protein tryosine phosphatase-like IA-2 (IA-2A) were measured by radiobinding assays. A short duration of total breast-feeding was associated with an increased risk of GADA and/or IAA above the ninety-fifth percentile at 5 years of age (OR 2.09, 95% CI 1.45, 3.02; P<0.000) as well as with an increased risk of IAA above the ninety-fifth percentile at this age (OR 2.89, 95% CI 1.81, 4.62, P<0.000). A short duration of exclusive breast-feeding was associated with an increased risk of GADA, IAA and/or IA-2A above the ninety-ninth percentile (OR 2.01, 95% CI 1.08, 3.73; P=0.028) as well as with an increased risk of IA-2A above the ninety-ninth percentile (OR 3.50, 95% CI 1.38, 8.92, P=0.009) at 5 years of age. An early introduction of formula was associated with an increased risk of GADA, IAA and/or IA-2A above the ninety-ninth percentile (OR 1.84, 95% CI 1.01, 3.37; P=0.047) at 5 years of age. The positive association between a short duration of both total and exclusive breast-feeding, as well as an early introduction of formula, and positivity for beta-cell autoantibodies in children from the general population suggest that breast-feeding modifies the risk of beta-cell autoimmunity, even years after finishing breast-feeding.