Vitamin D is linked to a number of adverse pregnancy outcomes through largely unknown mechanisms. This study was conducted to examine the role of vitamin D status in metabolomic profiles in a group of 30 pregnant, African American adolescents (17.1 + 1.1 years) at midgestation (26.8 + 2.8 weeks), in 15 adolescents with 25-hydroxy vitamin D (25(OH)D) !20 ng/mL, and in 15 teens with 25(OH)D <20 ng/mL. Serum metabolomic profiles were examined using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. A novel hierarchical mixture model was used to evaluate differences in metabolite profiles between low and high groups. A total of 326 compounds were identified and included in subsequent statistical analyses. Eleven metabolites had significantly different means between the 2 vitamin D groups, after correcting for multiple hypothesis testing: pyridoxate, bilirubin, xylose, and cholate were higher, and leukotrienes, 1,2-propanediol, azelate, undecanedioate, sebacate, inflammation associated complement component 3 peptide (HWESASXX), and piperine were lower in serum from adolescents with 25(OH)D !20 ng/mL. Lower maternal vitamin D status at midgestation impacted serum metabolic profiles in pregnant adolescents.