BACKGROUND: Late-onset sepsis (LOS) affects a large proportion of pre-term neonates in neonatal intensive care units (NICUs) worldwide, with a high morbidity and related mortality. Lactoferrin is a mammalian milk glycoprotein involved in innate immunity. Recent data show that bovine lactoferrin (bLF) prevents late-onset sepsis in preterm very low birth weight (VLBW) neonates. METHODS: This is a secondary analysis of data from a multicenter randomized controlled trial where preterm VLBW neonates randomly received bLF, bLF + Lactobacillus rhamnosus GG, or placebo. Her I analyze the incidence rates of the primary outcome late-onset sepsis, but also the secondary outcomes including incidence of urinary tract infections, fungal colonization, invasive fungal colonization, rate of progression from colonization to infection, necrotizing enterocolitis, threshold retinopathy of prematurity, severe intraventricular hemorrhage and bronchopulmonary dysplasia and adverse effects or intolerance in all groups. RESULTS: This study included 472 low birth weight infants whose clinical, nutritional, and demographical characteristics were similar. A statistically significant reduction in late-onset sepsis was observed in groups that received either lactoferrin alone (RR 0.34, 95% CI 0.17, 0.70; P=0,002) or in combination with Lactobacillus rhamnosus GG (RR 0.27, 95% CI 0.12, 0.60; P<0.001). In subgroup analyses, infants weighing less than 1000 g had significant reduction in late-onset sepsis after oral lactoferrin supplementation alone. Invasive fungal colonization was significantly decreased in the group receiving bLF (P=0.004). Prophylaxis with oral lactoferrin alone did not reduce the incidence of NEC (P=0.09), but there were a significant reduction in threshold retinopathy of prematurity in the group receiving oral lactoferrin alone (P=0.02). No adverse effects or intolerances occurred. CONCLUSIONS: Prophylactic oral administration of bLF reduces the incidence of late-onset sepsis in infants weighing less than 1500 g and most effective in infants weighing less than 1000 g. Oral lactoferrin prophylaxis also reduces the incidence of invasive fungal infection in preterm VLBW neonates and the incidence retinopathy of prematurity. Well-designed, randomized trials should address dosing, duration, type of lactoferrin (bovine or human) prophylaxis in prevention of sepsis and other invasive infections.