Secreted factors derived from Lactobacillus are able to dampen pro-inflammatory cytokine responses. Still, the nature of these components and the underlying mechanisms remain elusive. Here, we aimed to identify the components and the mechanism involved in the Lactobacillus-mediated modulation of immune cell activation. PBMC were stimulated in the presence of the cell free supernatants (CFS) of cultured Lactobacillus rhamnosus GG and Lactobacillus reuteri DSM 17938, followed by evaluation of cytokine responses. We show that lactobacilli-CFS effectively dampen induced IFN-γ and IL-17A responses from T- and NK cells in a monocyte dependent manner by a soluble factor. A proteomic array analysis highlighted Lactobacillus-induced IL-1 receptor antagonist (ra) as a potential candidate responsible for the IFN-γ dampening activity. Indeed, addition of recombinant IL-1ra to stimulated PBMC resulted in reduced IFN-γ production. Further characterization of the lactobacilli-CFS revealed the presence of extracellular membrane vesicles with a similar immune regulatory activity to that observed with the lactobacilli-CFS. In conclusion, we have shown that lactobacilli produce extracellular MVs, which are able to dampen pro-inflammatory cytokine responses in a monocyte-dependent manner.