Live probiotic bacteria are effective in reducing gut permeability and inflammation. We have previously shown that probiotics release peptide bioactive factors that modulate epithelial resistance in vitro. Aim: The objectives of this study were to determine the impact of factors released from Bifidobacteria infantis on intestinal epithelial cell permeability and tight junction proteins, and to assess whether these factors retain their bioactivity when administered to IL-10 deficient mice. Methods: B. infantis conditioned media (BiCM) was applied to T84 human epithelial cells in the presence and absence of TNF and IFN. Transepithelial resistance (TER), tight junction proteins (claudins 1, 2, 3, and 4, ZO-1, and occludin) and MAP kinase activity (p38 and ERK) were examined. Acute effects of BiCM on intestinal permeability were assessed in colons from IL-10 deficient mice in Ussing chambers. A separate group of IL-10 deficient mice was treated with BiCM for 30 days and then assessed for intestinal histological injury, cytokine levels, epithelial permeability, and immune response to bacterial antigens. Results: In T84 cells, BiCM increased TER, decreased claudin-2, and increased ZO-1 and occludin expression. This was associated with enhanced levels of phospho-ERK and decreased levels of phospho-p38. BiCM prevented TNF and IFN induced drops in TER and re-arrangement of tight junction proteins. Inhibition of ERK prevented the BiCM-induced increase in TER and attenuated the protection from TNF and IFN. Oral BiCM administration acutely reduced colonic permeability in mice while long-term BiCM treatment in IL-10 deficient mice attenuated inflammation, normalized colonic permeability, and decreased colonic and splenic IFN secretion. Conclusion: Peptide bioactive factors from Bifidobacteria infantis retain their biological activity in vivo and are effective in normalizing gut permeability and improving disease in an animal model of colitis. The effects of BiCM are mediated in part by changes in MAP kinases and tight junction proteins.