BACKGROUND: Recently, folic acid supplementation during pregnancy was implicated as a potential risk factor for atopic diseases in childhood. OBJECTIVE: To investigate whether folic acid supplementation and higher intracellular folic acid (ICF) levels during pregnancy increase the risk of childhood atopic diseases. METHODS: In the KOALA Birth Cohort Study (N = 2834), data on eczema and wheeze were collected by using repeated questionnaires at 3, 7, 12, and 24 months, 4 to 5 years, and 6 to 7 years after delivery. Atopic dermatitis and total and specific immunoglobulin E levels were determined at age 2 years and asthma and lung function at age 6 to 7 years. We defined folic acid use as stand-alone and/or multivitamin supplements according to the period of use before and/or during pregnancy. ICF levels were determined in blood samples taken at ∼35 weeks of pregnancy (n = 837). Multivariable logistic and linear regression analyses were conducted, with generalized estimating equation models for repeated outcomes. RESULTS: Maternal folic acid supplement use during pregnancy was not associated with increased risk of wheeze, lung function, asthma, or related atopic outcomes in the offspring. Maternal ICF level in late pregnancy was inversely associated with asthma risk at age 6 to 7 years in a dose-dependent manner (P for trend = .05). CONCLUSIONS: Our results do not confirm any meaningful association between folic acid supplement use during pregnancy and atopic diseases in the offspring. Higher ICF levels in pregnancy tended, at most, toward a small decreased risk for developing asthma.