Vitamin B 12 deficiency has been associated with increased risk of adverse pregnancy outcomes. Few prospective studies have investigated the burden or determinants of vitamin B 12 deficiency early in life, particularly among pregnant adolescents and their children. The objectives of this study were to determine the prevalence of vitamin B 12 deficiency and to examine associations between maternal and neonatal vitamin B 12 status in a cohort study of healthy pregnant adolescents. Serum vitamin B 12 and folate concentrations were measured in adolescents at mid-gestation ( n = 124; 26.4 ± 3.5 weeks) and delivery ( n = 131; 40.0 ± 1.3 weeks), and in neonates at birth using cord blood. Linear regression was used to examine associations between maternal and neonatal vitamin B 12 status. Although the prevalence of vitamin B 12 deficiency (<148.0 pmol/L; 1.6%) in adolescents was low during pregnancy, 22.6% of adolescents were vitamin B 12 insufficient (<221.0 pmol/L; 22.6%) at mid-gestation. Maternal vitamin B 12 concentrations significantly decreased from mid-gestation to delivery ( p < 0.0001), and 53.4% had insufficient vitamin B 12 status at delivery. Maternal vitamin B 12 concentrations ( p < 0.001) and vitamin B 12 deficiency ( p = 0.002) at delivery were significantly associated with infant vitamin B 12 concentrations in multivariate analyses, adjusting for gestational age, maternal age, parity, smoking status, relationship status, prenatal supplement use, pre-pregnancy body mass index, race, and intake of vitamin B 12 and folate. Maternal vitamin B 12 concentrations significantly decreased during pregnancy and predicted neonatal vitamin B 12 status in a cohort of healthy pregnant adolescents.