ABSTRACT: Findings on maternal 25-hydroxyvitamin D (25[OH]D) and neonatal anthropometry are inconsistent, and may at least be partly due to variations in gestational week (GW) of 25(OH)D measurement and the lack of longitudinal 25(OH)D measurements across gestation. The aim of the current study was to examine the associations of longitudinal measures of maternal 25(OH)D and neonatal anthropometry at birth. This study included 321 mother⁻offspring pairs enrolled in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Fetal Growth Studies⁻Singletons. This study was a prospective cohort design without supplementation and without data on dietary supplementation. Nevertheless, measurement of plasma 25(OH)D reflects vitamin D from different sources, including supplementation. Maternal concentrations of total 25(OH)D were measured at 10⁻14, 15⁻26, 23⁻31, and 33⁻39 GW and categorized as <50 nmol/L, 50⁻75 nmol/L, and >75 nmol/L. Generalized linear models were used to examine associations of 25(OH)D at each time-point with neonate birthweight z-score, length, and sum of skinfolds at birth. At 10⁻14 GW, 16.8% and 49.2% of women had 25(OH)D <50 nmol/L and between 50⁻75 nmol/L, respectively. The association of maternal 25(OH)D with neonatal anthropometry differed by GW and women's prepregnancy BMI (normal (<25.0 kg/m²), overweight/obese (25.0⁻44.9 kg/m²)). All analyses were stratified by prepregnancy BMI status. Among women with an overweight/obese BMI, 25(OH)D <50 nmol/L at 10⁻14 GW was associated with lower birthweight z-score (0.56; 95% CI: -0.99, -0.13) and length (-1.56 cm; 95% CI: -3.07, -0.06), and at 23⁻31 GW was associated with shorter length (-2.77 cm; 95% CI: -13.38, -4.98) and lower sum of skinfolds (-9.18 mm; 95% CI: -13.38, -4.98). Among women with a normal BMI, 25(OH)D <50 nmol/L at 10⁻14 GW was associated with lower sum of skinfolds (-2.64 mm; 95% CI: -5.03, -0.24), at 23⁻31 GW was associated with larger birthweight z-scores (0.64; 95% CI: 0.03, 1.25), and at 33-39 GW with both higher birthweight z-score (1.22; 95% CI: 0.71, 1.73) and longer length (1.94 cm; 95% CI: 0.37, 3.52). Maternal 25(OH)D status during pregnancy was associated with neonatal anthropometric measures, and the associations were specific to GW of 25(OH)D measurement and prepregnancy BMI.