Extended early antibiotic exposure in the neonatal intensive care unit is associated with an increased risk for the development of late-onset sepsis (LOS). However, few studies have examined the mechanisms involved. We sought to determine how the neonatal microbiome and intestinal immune response is altered by transient early empiric antibiotic exposure at birth. Neonatal mice were transiently exposed to broad-spectrum antibiotics from birth for either 3- (SE) or 7-days (LE) and were examined at 14-days-old. We found that mice exposed to either SE or LE showed persistent expansion of Proteobacteria (2 log difference, P < 0.01). Further, LE mice demonstrated baseline translocation of E. coli into the liver and spleen and were more susceptible K. pneumoniae-induced sepsis. LE mice had a significant and persistent decrease in type 3 innate lymphoid cells (ILC3) in the lamina propria. Reconstitution of the microbiome with mature microbiota by gavage in LE mice following antibiotic exposure resulted in an increase in ILC3 and partial rescue from LOS. We conclude that prolonged exposure to broad spectrum antibiotics in the neonatal period is associated with persistent alteration of the microbiome and innate immune response resulting in increased susceptibility to infection that may be partially rescued by microbiome reconstitution.