Propionibacterium freudenreichii is a Gram-positive, pleiomorphic, microaerophilic and probiotic bacterium with a long history of safe use, as a cheese starter and for the production of vitamin B12 and of organic acids. Its probiotics traits have been consistently associated with surface proteins and secreted metabolites, including antitumoral short-chain fatty acids, bifidogenic factors and immunomodulatory proteins. We previously reported that P. freudenreichii produced extracellular vesicles (EVs) as part of their health-promoting roles. EVs are nanosized membrane-encapsulated particles that play an essential role in communication between cells by their ability to transport bioactive molecules (proteins, nucleic acids, lipids, enzymes, toxins, metabolites) from a donor to a recipient cell. They act by transferring their content to target cells or by a specific interaction between ligands present on their surface and receptors expressed by target cells [1]. Recently, we showed that EVs produced by the probiobic P. freudenreichii strain CIRM-BIA129 (PF129) mimic the immunomodulatory properties of parent cells in vitro (i.e., modulating NF-κB transcription factor activity and LPS-induced IL-8 release), which underlies the role of EVs as mediators of the probiotic effects of the bacterium [2]. Here, we will present data on the mechanism of action and interaction between EVs derived from PF129 and human epithelial cells (HT-29) stimulated by pro-inflammatory LPS. PF129-derived EVs were purified by size-exclusion chromatography (SEC). The ability of EVs to interact with HT-29 cells and to be internalized was evaluated by confocal microscopy and flow cytometry. The impact of various incubation times, EV doses and inhibitors of the cellular internalization was then evaluated. Likewise, the expression of various TLR and immune genes was analyzed by RT-qPCR and the production of interleukin-8 (IL-8) was analyzed by enzyme-linked immunosorbent assay (ELISA). Notably, the ability of PF129-derived EVs to reduce the pro-inflammatory cytokine IL-8, modulate the expression of genes involved in the activation of NF-κB and immune genes in vitro will be presented as well as the ability of HT-29 cells to internalize PF129-derived EVs by endocytosis. Our findings point out that EVs might interact with and/or be internalized into the target cells by different pathways and then initiate a cell signaling cascades that acts on the inflammatory response. However, this internalization route into the target cells could be dependent of their size, membrane structure and EV content. This work contributes to demonstrate the promising future for bacterial-derived EVs as a biotherapeutic tool and a next-generation delivery platform for postbiotic applications