The clearance of apoptotic cells is an essential process to maintain homeostasis of immune system, which is regulated by immunoregulatory cytokines such as TGFβ. We show here that Extracellular Vesicles (EVs) were highly released from apoptotic cells, and contributed to macrophage production of TGFβ in vitro and in vivo. We further elucidated mechanistically that phosphatidylserine in EVs was a key triggering-factor, and transcription factor FOXO3 was a critical mediator for apoptotic EV-induced TGFβ in macrophages. Importantly, we found that macrophages pre-exposed to EVs exhibited an anti-inflammatory phenotype. More strikingly, administration of EVs in vivo promotes Tregs differentiation and suppresses Th1 cell response, and ameliorates experimental colitis. Thus, apoptotic-EV-based treatment might be a promising therapeutic approach for human autoimmune disease.