The prevalence of and mortality from non-tuberculous mycobacteria (NTM) infections have been steadily increasing worldwide. Most NTM infections are caused by Mycobacterium avium-intracellulare complex (MAC). MAC can escape from killing by neutrophils, which are professional phagocytes. However, the involvement of neutrophils in the pathogenesis of MAC infection is poorly understood. The present study assessed the roles of neutrophil extracellular trap (NET) formation in neutrophil defense mechanisms against infection with MAC strains, including M. avium isolated from patients with severe or mild lung tissue destruction. Although all MAC induced NET formation, non-pathogenic mycobacteria (M. gordonae and M. smegmatis) slightly but not significantly induced NET formation. Peptidylarginine deiminase 4 (PAD4) inhibitor reduced MAC-induced NET formation but did not affect MAC escape from neutrophils. PAD4 inhibition attenuated the MAC-induced matrix metalloproteinase (MMP)-8 and 9 release to the levels of MMPs from non-pathogenic mycobacteria. MAC also induced interleukin (IL)-8 release by neutrophils, a process independent of MAC-induced NET formation. Taken together, these findings suggest that MAC induce NET formation, IL-8 release and NETs-dependent release of MMP-8 and -9 from neutrophils, leading to neutrophil accumulation and further inflammation, thereby enhancing the progression of infection in the lungs.