Introduction and objectives Like almost all living cells, Staphylococcus aureus produced extracellular vesicles (EVs), which are composed of a lipid bilayer protecting a large number of bioactive molecules such as proteins, lipids, metabolites and nucleic acids. In the past years, there has been growing interest for EVs of bacteria due to their roles in infection and pathogenesis. Indeed, many studies demonstrate their involvement in cell-to-cell communication by transporting and transferring their cargo to recipient cells. EVs may have distinct activities, depending on the producing cell, their functional cargo and their mode of action in recipient cells. It appears that EVs must be central in host-pathogen interactions. However, researches on how pathogen derived-EVs act on host cells are still scarce. Materials and methods Our first objective was to provide a physico-biochemical characterization EVs produced by the methicillin-resistant Staphylococcus aureus strain N315. To mimic infection, N315 strain was grown in RPMI + 10% LB medium and N315-derived EVs were purified by size exclusion chromatography from culture supernatants. EV size and concentration were determined by nanoparticle tracking analysis. Western blot with anti-lipoteichoic acids and anti-peptidoglycan antibodies were performed on EVs lysate sample. Whole-cell and EVs proteome was identified by LC-MS. EVs was treated with RNAse A to remove external RNAs contaminant and the RNA content was analyzed with Bioanalyzer. The second objective was to evaluate impacts of EVs on the expression of several inflammatory genes, as well as their routes of entry into human non-phagocytic cells. EVs uptake by the human osteoblast-like MG- 63 non-phagocytic cell line was evaluated by confocal microscopy and flow cytometry in presence of various transport inhibitors. The impact of EVs on the expression of several MG-63 immune related genes was determined by RT-qPCR. Results, discussion and conclusion S. aureus N315 produced EVs of a typical spherical shape with an average size around 90 nm. Our results show that EVs contained lipoteichoic acid, peptidoglycan and ribonucleic acid. Proteomic comparison of whole cells and its derived EVs revealed the selective packing mechanisms of proteins into N315 EVs, notably of lipoproteins. MG-63 cells can internalize EVs in a dose- and time-dependent manner and via mainly dynamin-mediated endocytosis. EVs induced the expression of numerous immune and signaling genes such as IL-6, IL-8, MCP-1, IL1 , TLR2, TLR4 and TLR7. The expression of some them drastically decreased when EVs uptake was blocked, while others remained unchanged. Altogether, our results showed the ability of N315 EVs to trigger both extracellular and intracellular signaling pathway in host cells. This works shed light on new insights into staphylococcal pathogenesis and potentially provide new alternatives for the treatment and prevention of S. aureus infections.