Little is known about the metabolic turnover and excretion of vitamin K in healthy newborn infants and the metabolic consequences of prophylactic regimens designed to protect against vitamin K deficiency bleeding (VKDB). We measured the excretion of two urinary metabolites (≤24 h) of vitamin K (5C- and 7C-aglycones) in term infants before (n = 11) and after (n = 5) a 1000 μg i.m. dose of vitamin K1 (K1) and in preterm infants after 200 μg i.m. (n = 4), 500 μg i.m. (n = 4), or 200 μg i.v. (n = 5). In preterm infants, we also measured serum K1, vitamin K1 2,3-epoxide, and PIVKA-II at 5 d postpartum. Before prophylaxis, the rate of 5C- and 7C-aglycone excretion was 25 times lower than adults, reflecting low vitamin K stores at birth. After prophylaxis, the excretion rate correlated to K1 dose (r = 0.6) but was two orders of magnitude lower than that in adults, probably reflecting the immaturity of neonatal catabolism. All term and 10 of 13 preterm infants mainly excreted 5C-aglycone. We present evidence that increased excretion of the 7C-aglycone was associated with metabolic overload because of the exposure to high-tissue K1 concentrations. Measurement of the 5C- and 7C-aglycones may facilitate longitudinal studies of vitamin K status in neonates and aid the development of improved prophylactic regimens.