In dermatomyositis (DM), anti-melanoma differentiation-associated gene 5 (MDA5) autoantibody (autoAb) marks a subtype with low grade or absent muscle inflammation but frequent and rapidly progressive interstitial lung disease (ILD). The pathogenesis of ILD remains poorly unknown. The aim of the study is to explore whether neutrophil extracellular traps (NETs) are involved in the development of ILD in DM patients with anti-MDA5 autoAb. Patients with clinically amyopathic dermatomyositis (CADM, n = 20), classic dermatomyositis (cDM, n = 30), polymyositis (PM, n = 20), and healthy controls (HC, n = 20) were enrolled. Anti-MDA5 autoantibody and Krebs von den Lungen-6 (KL-6) were detected by ELISA. Circulating levels of NETs were assessed by the quantification of both serum cell-free DNA (cfDNA) and LL-37 (cathelicidin LL-37). Immunofluorescent staining was used to visualize NETs ex vivo. The elevated circulating NETs level was detected in DM patients with ILD complication. Compared to anti-MDA5 AbDM patients, anti-MDA5 AbDM patients had the higher concentrations of serum cfDNA (293 ± 69 vs 252 ± 63 ng/ml; P = 0.035) and serum LL-37 (0.6 ± 1.0 vs 0.2 ± 0.2 ng/ml; P = 0.026). Positive correlations were established between serum levels of cfDNA and KL-6 in DM patients (r = 0.4422, P = 0.0003). anti-MDA5 Absera, other than anti-MDA5 Absera, could induce greater numbers of normal neutrophils to form NETs in vitro. These data suggest that aberrant NETs formation may be involved in the pathogenesis of ILD in DM patients with anti-MDA5 autoAb.