jats:titleABSTRACT</jats:title> jats:sec jats:titleBACKGROUND</jats:title> jats:pVitamin D is important to maternal, fetal, and infant health, but quality data on vitamin D status in low- and middle-income countries and response to cholecalciferol supplementation in pregnancy are sparse.</jats:p> </jats:sec> jats:sec jats:titleOBJECTIVE</jats:title> jats:pWe characterized vitamin D status and vitamin D metabolite change across pregnancy and in response to cholecalciferol supplementation in rural Gambia.</jats:p> </jats:sec> jats:sec jats:titleMETHODS</jats:title> jats:pThis study was a secondary analysis of samples collected in a 4-arm trial of maternal nutritional supplementation [iron folic acid (FeFol); multiple micronutrients (MMN); protein energy (PE) as lipid-based supplement; PE + MMN]; MMN included 10 μg/d cholecalciferol. Plasma 25-hydroxycholecalciferol [25(OH)D3], 24,25-dihydroxycholecalciferol [24,25(OH)2D3], and C3-epimer-25-hydroxycholecalciferol [3-epi-25(OH)D3] were measured by LC-MS/MS in 863 women [aged 30 ± 7 y (mean ± SD)] in early pregnancy (presupplementation) and late pregnancy, (gestational age 14 ± 3 and 30 ± 1 wk). Changes in 25(OH)D3 and vitamin D metabolite concentrations and associations with pregnancy stage and maternal age and anthropometry were tested.</jats:p> </jats:sec> jats:sec jats:titleRESULTS</jats:title> jats:pEarly pregnancy 25(OH)D3 concentration was 70 ± 15 nmol/L and increased according to pregnancy stage (82 ± 18 and 87 ± 17 nmol/L in the FeFol and PE-arms) and to cholecalciferol supplementation (95 ± 19 and 90 ± 20 nmol/L in the MMN and PE + MMN-arms) (P &lt; 0.0001). There was no difference between supplemented groups. Early pregnancy 25(OH)D3 was positively associated with maternal age and gestational age. Change in 25(OH)D3 was negatively associated with late pregnancy, but not early pregnancy, triceps skinfold thickness. The pattern of change of 24,25(OH)2D3 mirrored that of 25(OH)D3 and appeared to flatten as pregnancy progressed, whereas 3-epi-25(OH)D3 concentration increased across pregnancy.</jats:p> </jats:sec> jats:sec jats:titleCONCLUSION</jats:title> jats:pThis study provides important data on the vitamin D status of a large cohort of healthy pregnant women in rural Africa. Without supplementation, vitamin D status increased during pregnancy, demonstrating that pregnancy stage should be considered when assessing vitamin D status. Nutritionally relevant cholecalciferol supplementation further increased vitamin D status. These data are relevant to the development of fortification and supplementation policies in pregnant women in West Africa.</jats:p> </jats:sec>