Bifidobacteria are gram positive anaerobic bacteria and are the predominant intestinal microorganism in breast-fed infants. Since previous studies have shown that Bifidobacteria supplementation in a newborn rat model reduced the incidence of NEC (18/26 control vs 5/22 Bifidobacteria-treated, p < 0.01), we sought to characterize the mechanisms responsible for this improved outcome. Newborn Sprague-Dawley rats were challenged with asphyxia (nitrogen for 50 seconds, twice daily) and formula feeding (artificial puppy formula, 200 kcal/kg/day via orogastric feeding tube q 3 hours), and some treated with exogenous Bifdiobacteria (109 organisms/animal q day) via an orogastric feeding tube to effect Bifidobacteria colonization as previously described. At various time intervals, animals were evaluated for plasma endotoxin concentration, mucosal permeability (via fluorescein-labeled dextran, mw 73,000, given orogastrically and plasma samples measured spectrophotofluorometrically), and intestinal phospholipase A2 gene expression (PLA2 mRNA measured using quantitative PCR). We found that the Bifidobacteria group had significantly less endotoxinemia than controls between 48 and 72 hours (21 ± 3 EU/ml Bifidobacteria-treated vs 191± EU/ml controls, p < 0.01). Furthermore, intestinal PLA2 gene expression was significantly lower in Bifidobacteria-treated animals at 48 hours (42.6 ±5.1 pg/μg RNA vs 735 ± 110 pg/μg RNA controls, p < 0.01). Alterations in mucosal permeability were similar between groups at all time points. We conclude that Bifidobacteria supplementation reduces NEC in part via the downregulation of the inflammatory cascade by reducing the endotoxin load and subsequent effect on PLA2. The role of Bifidobacteria in modulating intestinal disease warrants further study.