Introduction Propionibacterium freudenreichii, is a probiotic bacterium, with an established immunomodulatory role. Selected strains indeed induce the release of regulatory IL-10. Extractable surface proteins of the S-layer type (Slp) are involved in such modulation, but the exact mode of interaction with the host remains unknown. Mucositis is a common side effect of cytotoxic chemotherapy characterized by mucosal injury, inflammation, diarrhea, and weight loss. We hypothesized that immunomodulatory P. freudenreichii may mitigate inflammation and that SlpB may be required for such an effect. Extracellular vesicles (EVs) are nanometric spherical structures involved in intercellular communication, whose production is considered a widespread phenomenon in living organisms. Bacterial EVs are associated with several processes that include survival, competition, pathogenesis, and immunomodulation. We hypothesized that, in addition to surface exposure and secretion of proteins, P. freudenreichii may produce EVs and thus export immunomodulatory proteins to interact with the host. Methods P. freudenreichii CIRM-BIA 129, previously selected for its ability to induce IL-10 release by PBMCs, was administered orally to mice, prior to colitis induction using TNBS. The slpB gene was inactivated by insertional inactivation. Both parental wild type and mutant strains were administered orally to mice, prior to induction of mucositis using 5-FU. The SlpB protein was purified and its effect on cultured human intestinal epithelial cells investigated with respect to cytokines expression. EVs were purified from cell-free culture supernatants of the probiotic strain P. freudenreichii CIRM-BIA 129, prior to physicochemical and functional characterization. Results Consumption of P. freudenreichii CIM-BIA129 strain protected mice against TNBS-induced colitis in mice, alleviating severity of symptoms, modulating local and systemic inflammation, as well as colonic oxidative stress and epithelial cell damages. This strain prevented 5-FU-induced mucositis in mice, regulated key markers, including the expression of Claudin-1 (Cld1) and IL-17a (Il17a), as well as the levels of IL-12 and IL-1β cytokines. The slpB gene was mutated by insertional inactivation and the absence of SlpB protein in the resulting mutant was verified. The mutant strain displayed opposite immunomodulatory effect and failed to mitigate induced mucositis. In HT-29 human intestinal epithelial cells P. freudenreichii reduced expression of IL-8 and TNF-α cytokines in LPS-stimulated cells. P. freudenreichii ΔslpB, lacking the SlpB protein, failed to do so. In the same cells, purified SlpB was shown to induce expression of IL-10 in a dose-dependent manner. Extracellular vesicles (EVs), they were purified from cell-free culture supernatants of P. freudenreichii. They showed typical shapes and sizes of EVs and contained a broad range of proteins, including SlpB. EVs modulated inflammatory responses, IL-8 release and NF-κB activity, in HT-29 human intestinal epithelial cells, as well as the NF-κB pathway. Discussion This work emphasizes the importance of SlpB in immunomodulatory P. freudenreichii. It constitutes the first report on identification of P. freudenreichii-derived EVs, alongside their physicochemical, biochemical and functional characterization. It opens perspectives for the development of functional foods and other bioactive products in order to help preventing and/or treating inflammatory diseases, and to decrease side effects of chemotherapy.