Introduction and objectives Propionibacterium freudenreichii is a Gram-positive, pleiomorphic, microaerophilic and probiotic bacterium with anti-pathogenic activity, anticancer potential and immunomodulatory properties [1]. We previously reported that P. freudenreichii CIRM-BIA129 produced extracellular vesicles (EVs) as part of their health-promoting roles and modulated NFκB transcription factor activity and LPS-induced IL-8 release in vitro, which demonstrated the role of EVs as mediators of the probiotic effects of the bacterium [2]. 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 [3]. Our aim was to decipher the mechanism of interaction between EVs derived from P. freudenreichii CIRM-BIA129 (EV129) and intestinal epithelium cells (HT-29) stimulated by LPS. Particularly, the capability of EV129 to modulate the expression of key genes involved in the TLR4 dependent NFkB pathway and to be internalized was determined. Material and Methods EV129 were purified by size-exclusion chromatography (SEC). Their ability to interact with HT-29 cells and to be internalized were evaluated by flow cytometry and confocal microscopy. The impact of various incubation times, EV doses and inhibitors of the cellular internalization were then evaluated. Effect of EV129 on the expression of HT-29 genes involved in the TLR4 dependent NFkB pathway was analyzed by RT-qPCR and the production of interleukin-8 (IL-8) by enzyme-linked immunosorbent assay (ELISA). Results, discussion and conclusion Our findings point out that EV129 are internalized by HT-29 cells through of dynamin-dependent endocytosis mechanism which is dependent of the GTPase dynamin such the activation of TLR4 [4]. Our study demonstrated that EV129 downregulated Tlr4 gene expression initiating a signal transduction intracellular which caused a cascade effect, leading to a reduction of NFkB and the pro-inflammatory cytokines IL8, IL1b and Tnf gene expression into HT-29 cells. This work contributes to demonstrate the promising future for the use of EV129 as a biotherapeutic tool in intestinal inflammatory disease.