Very low birthweight, preterm infants are at high risk of vitamin D deficiency at birth, which can persist during the first month of life. Vitamin D metabolite profiles of neonates represent a topic that is not well understood. This study aimed to investigate vitamin D metabolism in a cohort of 90 preterm infants (<1000g & <28 weeks gestational age [GA]) by measuring serum vitamin D metabolites during their NICU admission, including at birth (baseline, BL), 1 month after birth, and 36 weeks post-menstrual age (PMA) using liquid chromatography tandem mass spectrometry (LC-MS/MS). Specifically, we measured 25-OH-D, 24,25-(OH)2D3, 3-epi-25-OH-D3 and calculated the 25-OH-D3:24,25-(OH)2D3 ratio (R) as well as % of 3-epi-25-OH-D3; which we used to characterize vitamin D status, frequency of vitamin D deficiency and the extent of 3-epimerization and 24-hydroxylation of 25-OH-D3. To assess potential changes in vitamin D metabolism with maturity, a subgroup of neonates at <25 weeks and ≥25 weeks GA was explored for differences in vitamin D metabolites. The mean GA of the infants was 25±2 (SD), birth weight 782±206, 38% were female. Mean BL 25-OH-D was 19±12 ng/mL, with 62/90 infants exhibiting 25-OH-D < 20 ng/mL. At 1 month after birth and 36 weeks PMA, 25-OH-D increased to 51±28 and 56±29 ng/mL, with only 2 or 3 infants exhibiting 25-OH-D < 20 ng/mL at at 1 month and 36 weeks. BL 3-epi-25-OH-D3 was 3±2 ng/mL corresponding to 15% of total 25-OH-D3+3-epi-25-OH-D3; increasing to 57±37 ng/mL (50%) and 43±21 ng/mL (44%) at 1 month after birth and 36 weeks PMA. This dramatic increase 3-epimerization is consistent with other studies in neonates. While 24,25-(OH)2D3 appeared appropriate for 25-OH-D3 concentration at BL (1.1±0.9ng/mL; ratio(R) of 25-OH-D3:24,25-(OH)2D3=19); concentrations of 24,25-(OH)2D3 relative to 25-OH-D3 were lower at 1 month after birth (1.3±1.0 ng/mL; R=43) and 36 weeks PMA (2.2±0.8 ng/mL; R=29) as compared with BL, suggesting supressed CYP24A1 enzyme activity. Infants <25 weeks had a slightly lower R of 17.6 than ≥25 weeks, (R=20.3, p=0.015). All other metabolite measurements did not differ between the subgroups. Vitamin D deficiency, increased 3-epimerization and decreased 24-hydroxylation are a triad of metabolic aberrations that occur in very low birthweight infants until 36 weeks PMA. Vitamin D supplementation may be able to correct vitamin D deficiency. However, the physiological role of altered 3-epimerization and 24-hydroxylation during a period of rapid skeletal development remains unknown. Impact of these metabolic alterations on 1,25-(OH)2D3 concentrations continues to be investigated.