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Background: Vitamin D supplementation is recommended for breastfed infants. Maternal supplementation beginning in gestation is a potential alternative, but its efficacy in maintaining infant 25-hydroxyvitamin D [25(OH)D] concentration after birth is unknown. Objectives: We determined the effect of 3 doses of maternal vitamin D supplementation beginning in gestation and continued in lactation on infant serum 25(OH)D and compared the prevalence of infant serum 25(OH)D cutoffs (>30, >40, >50, and >75 nmol/L) by dose at 8 wk of age. Design: Pregnant women (n = 226) were randomly allocated to receive 10, 25, or 50 µg vitamin D3/d from 13 to 24 wk of gestation until 8 wk postpartum, with no infant supplementation. Mother and infant blood was collected at 8 wk postpartum. Results: At 8 wk postpartum, mean [nmol/L (95% CI)] infant 25(OH)D at 8 wk was higher in the 50-µg/d [75 (67, 83)] than in the 25-µg/d [52 (45, 58)] or 10-µg/d [45 (38, 52)] vitamin D groups (P < 0.05). Fewer infants born to mothers in the 50-µg/d group had a 25(OH)D concentration,30 nmol/L (indicative of deficiency) than infants in the 25- and 10-µg/d groups, respectively (2% compared with 16% and 43%; P< 0.05). Fewer than 15% of infants in the 10-or 25-µg/d groups achieved a 25(OH)D concentration .75 nmol/L compared with 44% in the 50-µg/d group (P < 0.05). Almost all infants (w98%, n = 44) born to mothers in the 50-µg/d group achieved a 25(OH)D concentration .30 nmol/L. At 8 wk postpartum, mean maternal 25(OH)D concentration was higher in the 50-µg/d [88 (84, 91)] than in the 25-µg/d [78 (74, 81)] or 10-µg/d [69 (66, 73)] groups (P< 0.05). Conclusions: Maternal supplementation beginning in gestation with 50 µg vitamin D3/d protects 98% of unsupplemented breastfed infants against 25(OH)D deficiency (<30 nmol/L) to at least 8 wk, whereas 10 or 25 µg vitamin D/d protects only 57% and 84% of infants, respectively. This trial was registered at clinicaltrials.gov as NCT01112891. [ABSTRACT FROM AUTHOR]