Objective This study was performed to detect the expression of NADPH, ROS, NOX2 and NOX4 in the skeletal muscle tissues of experimental autoimmune myositis mice and investigate the role and significance of reactive oxygen species mediated by NADPH oxidases in the pathogenesis of Idiopathic Inflammatory Myopathy. Methods Fourteen healthy female BALB/c mice were randomly divided into control group and EAM group (6 in control group; 8 in EAM group), and guinea pig skeletal muscle homogenate was used to induce experimental autoimmune myositis in EAM group. We should observe the changes of weight and clinical manifestations and measure the serum creatinase level, limb muscle strength and skeletal muscle pathology of mice in each group. Then the contents of NADPH and ROS in mice skeletal muscle tissues of each group were detected by spectrophotometry and ELISA respectively, and the expressions of NOX2 and NOX4 were measured by immunohistochemistry. Results The results of HE staining showed that the size, morphology and structure of skeletal muscle fibers in the control group were basically normal, with a small amount of inflammatory cell infiltration, and no muscle fibers atrophy, degeneration or necrosis. Skeletal muscle fibers of mice in the EAM group were different in size and thickness, with a large number of inflammatory cell infiltration and different degrees of atrophy, degeneration and necrosis. The histopathological scores of skeletal muscle of mice in the EAM group were significantly higher than that in the control group (P＜0.05). Compared with the control group, the levels of NADPH were decreased (P＜0.05) and levels of ROS were increased (P＜0.05) in the skeletal muscle tissues of the EAM group. The expressions of NOX2 and NOX4 in skeletal muscle tissues of EAM group mice were higher than that of control group mice (P＜0.05). The contents of ROS in skeletal muscle tissues of mice in EAM group were positively correlated with the Immunohistochemical scores of NOX2 and NOX4 (P＜0.05), and negatively correlated with the contents of NADPH (P＜0.05). The pathological scores of HE staining in skeletal muscle tissues of mice in EAM group were positively correlated with the Immunohistochemical scores of NOX2 and NOX4 and the contents of ROS (P＜0.05), and negatively correlated with the contents of NADPH (P＜0.05). Conclusion NOX2 and NOX4 may induce ferroptosis by consuming NADPH and producing a large amount of ROS, and ferroptosis may be involved in the pathogenesis of IIM by promoting oxidative damage and accelerating inflammatory response. NADPH, NOX2, NOX4 and ROS may be new indicators to evaluate the degree of muscle tissues injury in IIM.