Necrotizing enterocolitis (NEC) is associated with a high morbidity and mortality in very low birth weight infants. Several hypotheses regarding the pathogenesis of NEC have been proposed but to date no effective treatment is available. Previous studies suggest that probiotic supplementation is protective. We recently reported that probiotic (Bifidobacterium infantis) conditioned media (PCM) has an anti-inflammatory effect in cultured fetal human intestinal cells (H4) and fetal intestine explants. In this study, we tested in vivo if PCM protects neonatal mice from developing intestinal inflammation induced by exposure to Cronobacter sakazakii (C. sakazakii), an opportunistic pathogen associated with NEC. We found that infected neonatal mice had a significantly lower body weight than control groups. Infection led to ileal tissue damage including villous rupture, disruption of epithelial cell alignment, intestinal inflammation, apoptotic cell loss and decreased mucus production. Pre-treatment with PCM prevented infection decrease in body weight, attenuated enterocyte apoptotic cell death, mitigated reduced mucin production and maintained ileal structural . Infected ileum expressed reduced levels of IBα which could be restored upon pretreatment with PCM. We also observed a nuclear translocation of NF-B p65 in H4 cells exposed to, which was prevented in PCM pretreated cells. Finally, treatment of neonatal mice with PCM prior to infection sustained the capacity of ileal epithelial proliferation. This suggests that an active component(s) released into the culture media by Bifidobacterium infantis may prevent ileal damage by a pathogen linked to NEC.