Propionibacterium freudenreichii is a Gram-positive, pleiomorphic, microaerophilic and probiotic bacterium with long-dated use as cheese starter, for the production of vitamin B12 and organic acids. Its probiotics traits have been consistently associated with surface or 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) induced by LPS. Notably, the ability of PF129-derived EVs to modulate the expression of various TLR and immune genes in vitro will be presented as well as the ability of HT-29 cells to internalize PF129-derived EVs by dynamin-dependent 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 immune system. This work contributes to demonstrate the promising future for bacterial-derived EVs as a biotherapeutic tool and a next-generation delivery platform for probiotic applications.