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OBJECTIVE Vitamin D deficiency in pregnancy is associated with an increased risk of gestational diabetes mellitus (GDM) and neonatal vitamin D deficiency. We conducted a double-blind, randomized controlled trial of low-dose (LD) versus high-dose (HD) vitamin D supplementation to investigate the effects of vitamin D supplementation on glucose metabolism during pregnancy. RESEARCH DESIGN AND METHODS Women with plasma 25-hydroxyvitamin D (25OHD) levels <32 ng/mL before 20 weeks' gestation were randomized to oral vitamin D3 at 5,000 IU daily (HD) (n = 89) or the recommended pregnancy dose of 400 IU daily (LD) (n = 90) until delivery. The primary end point was maternal glucose levels on oral glucose tolerance test (OGTT) at 26-28 weeks' gestation. Secondary end points included neonatal 25OHD, obstetric and other neonatal outcomes, and maternal homeostasis model assessment of insulin resistance. Analysis was by intention to treat. RESULTS There was no difference in maternal glucose levels on OGTT. Twelve LD women (13%) developed GDM versus seven (8%) HD women (P = 0.25). Neonatal cord 25OHD was higher in HD offspring (46 ± 11 vs. 29 ± 12 ng/mL, P < 0.001), and deficiency was more common in LD offspring (24 vs. 10%, P = 0.06). Post hoc analysis in LD women showed an inverse relationship between pretreatment 25OHD and both fasting and 2-h blood glucose level on OGTT (both P < 0.001). Baseline 25OHD remained an independent predictor after multiple regression analysis. CONCLUSIONS HD vitamin D supplementation commencing at a mean of 14 weeks' gestation does not improve glucose levels in pregnancy. However, in women with baseline levels <32 ng/mL, 5,000 IU per day was well tolerated and highly effective at preventing neonatal vitamin D deficiency. [ABSTRACT FROM AUTHOR]