Background: Folate and cobalamin status changes markedly during infancy. Objective: We aimed to examine the influence of breastfeeding on folate and cobalamin status in healthy infants. Design: In a longitudinal study, we measured serum folate, cobalamin, holotranscobalamin, holohaptocorrin, methylmalonic acid, and homocysteine at birth and at ages 6, 12, and 24 mo (n ҃ 361, 262, 244, and 224, respectively). Breastfeeding status and nutrient intake were assessed by using questionnaires and 7-d weighed-food records (at 12 mo). Results: All indexes changed significantly from birth to age 24 mo (P 0.001). Folate was high until age 6 mo and then declined. At age 6 mo, folate was positively correlated with duration of exclusive breastfeeding ( ҃ 0.29; P 0.001). Cobalamin status declined after birth in breastfed but increased in nonbreastfed infants. Thus, holotranscobalamin (pmol/L) was lower in breastfed than in nonbreastfed children at age 6 mo [geometric x : 37 (95% CI: 33, 40) and 74 (64, 86), respectively], at 12 mo [51 (46, 56) and 76 (70, 82), respectively], and at 24 mo [65 (50, 83) and 90 (85, 97), respectively; P 0.05 for all]. Complementary feeding did not increase (6 mo) or modestly increased (12 mo) cobalamin status in breastfed children. At 12 mo, cobalamin intake (g/d), excluding breast milk cobalamin, was lower in breastfed than in nonbreastfed infants [geometric x : 1.4 (1.3, 1.6) and 2.4 (2.1, 2.6), respectively; P 0.001]. However, after adjustment for total cobalamin intake, cobalamin status (ie, holotranscobalamin) remained significantly lower in breastfed than in nonbreastfed infants [54 (49, 59) and 70 (64, 78), respectively; P 0.001]. Conclusions: Low cobalamin status is a characteristic finding in breastfed children. Reference limits according to age and breastfeeding status should be considered in early childhood.