Background: Neonatal vitamin K stores are precarious. Without adequate vitamin K prophylaxis preterm infants may be at particular risk of vitamin K deficiency bleeding (VKDB), but the optimal dose and route is unclear. Objective: To compare the vitamin K status of preterm infants during the first week of life and when on full enteral feeds, following three regimes of vitamin K prophylaxis after delivery. Design/Methods: Infants born at < 32 weeks gestation were randomised to receive vitamin K1 0.5 mg intramuscularly (IM) (group 1: control), 0.2 mg IM (group 2) or 0.2 mg intravenously (IV) (group 3) on day 1. Cord blood was obtained; venous blood samples at 5 days postnatal and 2 weeks after establishment of full enteral feeds were analysed for serum vitamin K1, vitamin K1 2,3-epoxide, descarboxyprothrombin (PIVKA-II), and prothrombin time. Results: Of 98 infants enrolled, 90 had a day 5 sample and 80 had a second sample obtained at a median (IQR) of 25 (22–31) days. Baseline characteristics (mean ±SD) for groups 1 (n=31), 2 (n=34) and 3 (n=33) were respectively: gestational age 28.3 ±2.5, 28.6 ± 2.3, and 28.1 ±2.6 weeks; birthweight 1025 ±379, 1138 ±379, and 1060 ±371 g. Serum vitamin K concentrations (ng/mL) Compared with the control group, day 5 vitamin K concentrations were significantly lower in group 2 (p = 0.04), and at the time of established feeds they were lower in group 3 (p = 0.03). Three infants (one in group 2; two in group 3) had undetectable levels of vitamin K at the time of the second sample, however in each case PIVKA-II was undetected. Eleven of ninety (12%) infants (seven in group 1; four in group 3) had detectable concentrations of vitamin K epoxide on day 5 (p = 0.007). Conclusions: Preterm infants given 0.2 mg or 0.5 mg vitamin K1 at birth have very high serum concentrations during the first week of life. The presence of vitamin K epoxide is significantly associated with a higher dose (0.5 mg) of vitamin K given IM and with a reduced dose (0.2 mg) given IV, and may reflect vitamin K overload of the immature liver by these regimes of prophylaxis. With a reduced dose given IV or IM, vitamin K can fall to undetectable levels by as early as the fourth postnatal week. The risk of subsequent late VKDB may be increased in these infants unless further vitamin K supplements are given.