Background Evidence linking prenatal maternal vitamin D supplementation and offspring risk of atopic eczema is inconsistent, with most data coming from observational studies. Methods Within the UK Maternal Vitamin D Osteoporosis Study (MAVIDOS) double-blind, randomised, placebo-controlled trial, we examined the relation of maternal vitamin D supplementation during pregnancy with offspring atopic eczema at ages 12, 24 and 48 months. In MAVIDOS, pregnant women were allocated to either cholecalciferol 1000 IU/day or matched placebo, taken from around 14 weeks’ gestation until delivery, with the primary outcome of neonatal whole-body bone mineral content. The prevalence of atopic eczema in the offspring was ascertained at ages 12 (n=636), 24 (n=611) and 48 (n=450) months, based on the UK Working Party Criteria for the Definition of Atopic Dermatitis. Results Mothers and offspring characteristics were similar between the intervention and placebo groups, apart from longer breastfeeding duration in the intervention group. Adjusting for breastfeeding duration, offspring of mothers who received 1000 IU cholecalciferol daily had a lower odds ratio (OR) of atopic eczema at age 12 months: OR (95%CI) 0.55 (0.32-0.97), p=0.04. The ORs of atopic eczema in the intervention group at ages 24 and 48 months were 0.77 (0.47-1.24) and 0.71 (0.35-1.43), respectively. Conclusion Our data demonstrated a clinically important reduction in offspring risk of atopic eczema in infancy following maternal cholecalciferol supplementation during pregnancy. The findings support a developmental influence on infantile atopic eczema and point to gestational cholecalciferol supplementation as a preventive strategy to reduce the burden of atopic eczema during infancy.