Background: Vitamin D is obtained from dietary sources and synthesized in the skin during exposure to ultraviolet B radiation in sunlight. During pregnancy, vitamin D is transported from mother to fetus through the placenta in the form of 25-hydroxyvitamin D [25(OH)D]. There is evidence that vitamin D influences neuronal differentiation, endocrine functions, and fetal brain growth. Animal studies indicate alterations in the offspring brain as a consequence of vitamin D deficiency during pregnancy. In humans, maternal vitamin D insufficiency has been linked to impaired child language development. Using data from a prebirth cohort with up to 22 years of follow-up, we examined the association of vitamin D status with proxies of offspring neurodevelopmental outcomes. During 1988-1989, pregnant women were recruited for the DaFO88 cohort (n = 965) in Aarhus, Denmark. Maternal concentrations of 25(OH)D were quantified in serum from week 30 of gestation via the LC-MS/MS method (n = 850). Offspring were followed up through national registries until the age of 22 years. We evaluated the association of the maternal concentration of 25(OH)D with offspring neurodevelopmental outcomes defined as first admission diagnosis or prescription of medication for (1) ADHD, (2) depression, and (3) scholastic achievement based on the mean grade on standardized written examinations in the 9th grade (final exams after 10 years of compulsory school in Denmark). Key Messages: Maternal concentrations of 25(OH)D were higher compared to current levels (median 76 nmol/l; 5th to 95th percentiles 23-152). There was a direct association between maternal vitamin D status and offspring depression (p_trend = 0.01); for ADHD there was no association. Scholastic achievement was slightly higher for offspring of mothers with a vitamin D status in the range of >50-125 nmol/l, but this nonlinear association was not statistically significant. Conclusions: Our analyses based on biomarker measurement of 25(OH)D from a cohort of 850 pregnant women combined with long-term follow-up showed no support for a beneficial fetal programming effect of vitamin D status with regard to behavioral and affective disorders and scholastic achievement.