Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The aim of this study was to evaluate the effects of Bifidobacterium bifidum treatment on development of NEC, cytokine regulation, and intestinal integrity in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), hand fed with formula (NEC), or hand fed with formula supplemented with 5 x 10(6) CFU B. bifidum per day (B. bifidum). All groups were exposed to asphyxia and cold stress to develop NEC. Intestinal injury, mucin and trefoil factor 3 (Tff3) production, cytokine levels, and composition of tight junction (TJ) and adherens junction (AJ) proteins were evaluated in the terminal ileum. B. bifidum decreased the incidence of NEC from 57 to 17%. Increased levels of IL-6, mucin-3, and Tff3 in the ileum of NEC rats was normalized in B. bifidum treated rats. Reduced mucin-2 production in the NEC rats was not affected by B. bifidum. Administration of B. bifidum normalized the expression and localization of TJ and AJ proteins in the ileum compared with animals with NEC. In conclusion, administration of B. bifidum protects against NEC in the neonatal rat model. This protective effect is associated with reduction of inflammatory reaction in the ileum, regulation of main components of mucus layer, and improvement of intestinal integrity.