Background/Objectives Vitamin D deficiency is common among pregnant women and since the fetus relies exclusively on maternal supply, deficiency could potentially interfere with fetal development. Vitamin D blood concentrations during pregnancy have been associated with offspring cardio- metabolic health in a few previous studies but the evidence is still inconsistent and only one previous study has followed the offspring into adulthood. The aim of the present study was to investigate the association between maternal serum concentration of Vitamin D (25 (OH)D) in week 30 of gestation and offspring cardio-metabolic risk factors at 20 years. Subjects/Methods A follow up study of a Danish birth cohort from 1988±89 (n = 965) was conducted. A blood sample was drawn from the women in week 30 of gestation. In 2008±2009, 95% of the original mother and child dyads could be identified in the central registration registry and were alive and living in Denmark. The offspring were followed up with self-reported anthropometrics (N = 629, 69%) and a clinical examination (N = 410, 45%). Multiple linear regression was used to estimate the association between maternal 25(OH)D and offspring cardio-metabolic risk factors adjusting for potential confounders. Results No overall association was observed between maternal 25(OH)D in week 30 of gestation and offspring cardio-metabolic risk factors. However, the analyses did suggest a possible inverse association with blood pressure in females. Conclusions No clear association between maternal 25(OH)D concentration in week 30 of gestation and cardio-metabolic risk factors in the 20 year old offspring was found.