(), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria-bacteria and bacteria-host interactions during infections. It is still unclear how EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from and mutant strains deficient in either prelipoprotein lipidation (Δ) or major surface proteins (Δ). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δ showed no stimulation. On the other hand, EVs isolated from the Δ mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.