Neutrophil extracellular trap (NET) formation plays an important role in inflammatory diseases. Although it is known that NET formation occurs via NADPH oxidase (NOX)-dependent and NOX-independent pathways, the detailed mechanism remains unknown. Therefore, in this study, we aimed to elucidate the role of mitochondria in NOX-dependent and NOX-independent NET formation. We generated mitochondrial DNA-deficient cells (ρ⁰ cells) by treating HL-60 cells with dideoxycytidine and differentiated them to neutrophil-like cells. These neutrophil-like ρ⁰ cells showed markedly reduced NOX-independent NET formation but not NOX-dependent NET formation. However, NET-associated intracellular histone citrullination was not inhibited in ρ⁰ cells. Furthermore, cells membrane disruption in NOX-dependent NET formation occurred in a Myeloperoxidase (MPO) and mixed lineage kinase domain like pseudokinase (MLKL)-dependent manner; however, cell membrane disruption in NOX-independent NET formation partially occurred in an MLKL-dependent manner. These results highlight the importance of mitochondria in NOX-independent NET formation.