Vitamin B12 deficiency has been associated with increased risk of adverse pregnancy outcomes. Few prospective studies have investigated the burden or determinants of vitamin B12 deficiency early in life, particularly among pregnant adolescents and their children. The objectives of this study were to determine the prevalence of vitamin B12 deficiency and to examine associations between maternal and neonatal vitamin B12 status in a cohort study of healthy pregnant adolescents. Serum vitamin B12 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 B12 status. Although the prevalence of vitamin B12 deficiency (<148.0 pmol/L; 1.6%) in adolescents was low during pregnancy, 22.6% of adolescents were vitamin B12 insufficient (<221.0 pmol/L; 22.6%) at mid-gestation. Maternal vitamin B12 concentrations significantly decreased from mid-gestation to delivery (p < 0.0001), and 53.4% had insufficient vitamin B12 status at delivery. Maternal vitamin B12 concentrations (p < 0.001) and vitamin B12 deficiency (p = 0.002) at delivery were significantly associated with infant vitamin B12 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 B12 and folate. Maternal vitamin B12 concentrations significantly decreased during pregnancy and predicted neonatal vitamin B12 status in a cohort of healthy pregnant adolescents.