Recently, many Gram-positive bacteria as well as Gram-negative bacteria have been reported to produce membrane vesicles (MVs), but little is known regarding the regulators involved in MV formation. We found that a Gram-positive anaerobic pathogen, Clostridium perfringens, produces MVs predominantly containing membrane proteins and cell wall components. These MVs stimulated proinflammatory cytokine production in mouse macrophage-like cells. We suggested that MVs induced interleukin-6 production through the Toll-like receptor 2 (TLR2) signaling pathway. Thus, the MV could have a role in the bacterium-host interaction and bacterial infection pathogenesis. Moreover, we found that the sporulation master regulator gene spo0A was required for vesiculogenesis. A conserved, phosphorylated aspartate residue of Spo0A was indispensable for MV production, suggesting that the phosphorylation of Spo0A triggers MV production. Multiple orphan sensor kinases necessary for sporulation were also required to maximize MV production. These findings imply that C. perfringens actively produces immunoactive MVs in response to the environment changing, as recognized by membrane-spanning sensor kinases and by modulating the phosphorylation level of Spo0A.