The growing incidence of human diseases involving inflammation and increased gut permeability makes the quest for protective functional foods more crucial than ever. Propionibacterium freudenreichii (P. freudenreichii) is a beneficial bacterium used in the dairy and probiotic industries. Selected strains exert anti-inflammatory effects, and the present work addresses whether the P. freudenreichii CIRM-BIA129, consumed daily in a preventive way, could protect mice from acute colitis induced by dextran sodium sulfate (DSS), and more precisely whether it could protect from intestinal epithelial breakdown induced by inflammation. P. freudenreichii CIRM-BIA129 mitigated colitis severity and inhibited DSS-induced permeability. It limited crypt length reduction and promoted the expression of Zonula Occludens-1 (ZO-1), without reducing interleukin-1beta mRNA (il-1ß) expression. In vitro, P. freudenreichii CIRM-BIA129 prevented the disruption of a Caco-2 monolayer induced by pro-inflammatory cytokines. It increased transepithelial electrical resistance (TEER) and inhibited permeability induced by inflammation, along with an increased ZO-1 expression. Extracellular vesicles (EVs) from P. freudenreichii CIRM-BIA129, carrying the surface layer protein (SlpB), reproduced the protective effect of P. freudenreichii CIRM-BIA129. A mutant strain deleted for slpB (ΔslpB), or EVs from this mutant strain, had lost their protective effects, and worsened both DSS-induced colitis and inflammation in vivo. These results shown that P. freudenreichii CIRM-BIA129 daily consumption has the potential to greatly alleviate colitis symptomss and, particularly, to counter intestinal epithelial permeability induced by inflammation by restoring ZO-1 expression through mechanisms involving S-layer protein B. They open new avenues for the use of probiotic dairy propionibacteria and/or postbiotic fractions thereof, in the context of gut permeability. NEW : 1. P.freudenreichii reduces DSS-induced intestinal permeability in vivo 2. P.freudenreichii does not inhibit inflammation but damages linked to inflammation 3. P.freudenreichii inhibits intestinal epithelial breakdown through S-layer protein B NOTEWORTHY : 1. The protective effects of P.freudenreichii depends on S-layer protein B 2. Extracellular vesicles from P. freudenreichii CB 129 mimic the protective effect of the probiotic ownloaded from journals.physiology.org/journal/ajpgi at INRAE Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (147.100.179.233) on