Preterm birth, bacterial colonization and formula feeding predispose to necrotizing enterocolitis (NEC). Antibiotics are commonly administered to prevent sepsis in preterm infants, but it is not known whether this affects intestinal immunity and NEC resistance. We hypothesized that broad-spectrum antibiotics treatment improves NEC resistance and intestinal structure, function and immunity in neonates. Caesarean-delivered preterm pigs were fed 3 d of parenteral nutrition followed by 2 d of enteral formula. Immediately after birth they were assigned to receive either antibiotics (oral and parenteral doses of gentamycin, ampicillin and metronidazole, ANTI, n=11) or saline in the control group (CON, n=13), given twice daily. NEC-lesions and intestinal structure, function, microbiology and immunity markers were recorded. None of the ANTI but 85% of the CON pigs developed NEC lesions by d 5 (0/11 vs. 11/13, P<0.05). ANTI pigs had higher intestinal villi (+60%), digestive enzyme activities (+53-73%), goblet cell densities (+110%), and lower myeloperoxidase (-51%) and colonic microbial density (105 vs. 1010 CFU, all p<0.05). Microarray transcriptomics showed strong down-regulation of genes related to inflammation and innate immune response to microbiota and marked up-regulation of genes related to amino acid metabolism, in particular threonine, glucose transport systems and cell cycle in 5 d-old ANTI pigs. In a follow-up experiment, 5 d of antibiotics prevented NEC at least until day 10. Neonatal prophylactic antibiotics effectively reduced gut bacterial load, prevented NEC, intestinal atrophy, dysfunction and inflammation, and enhanced expression of genes related to gut metabolism and immunity in preterm pigs.