Mycobacterium avium complex (MAC) is an ubiquitous acid-fast bacterium. MAC cell wall and membrane release extracellular vesicles (EVs) into different media. The immunogenic effects of EVs isolated from MAC remain unknown. The aim of this study was to determine the EVs effect on macrophage cytokine production. MAC EVs were extracted and purified using differential centrifuges also known as Claassen’s method, with some modifications. After protein analysis of EVs, and scanning electron microscopy (SEM), the EVs were injected into BALB/c mice for in vivo experiments. The concentration of interferon gamma (IFN-γ) and interleukin 10 (IL-10) in the spleen immune cell culture was measured by sandwich ELISA. We showed for the first time, that MAC can naturally produce EVs. The extraction method was technically-feasible, efficient and affordable. The SEM analysis showed that EVs diameter was similar to other studies on mycobacteria, and EVs maintained their spatial characterization. The results of the cytokine assays indicated that EV-treated cells secreted IL-10 (P = 0.034) but not IFN-γ (P = 0.037). Our findings suggest that EVs of M. avium could have anti-inflammatory effects. They can be used as a suppressor or regulator of inflammation via IL-10. The replication of the anti-inflammatory response of MAC EVs by future studies, may indicate a new therapeutic agent for inflammation.