Mutations in CYBB, encoding gp91phox subunit of NADPH oxidase in phagocytes, impair the respiratory burst of neutrophils and result in X-linked chronic granulomatous disease (CGD). While inflammatory response and NETosis are important modalities employed by neutrophils for pathogen clearance, variants in these cell functions in CGD neutrophils (CGD-PMN) could possibly explain the insufficient defense and accumulation of phagocytes in the sites of infection. To decipher the intrinsic features of CGD-PMN, neutrophils from X-linked CGD patients with CYBB mutations and age-matched healthy donors (HD-PMN) were compared. Our study found an enhanced spontaneous neutrophil extracellular trap (NET) formation with histone hypercitrullination in resting CGD-PMN. RNA sequencing (RNA-seq) analysis and qPCR validation further revealed a prominent activated type I interferon (IFN) gene signature. This suggested that in CGD patients an inefficient clearance of pathogen caused a chronic stimulation to provoke a spontaneous type I IFN-mediated neutrophil activation and NET formation which were never seen in resting neutrophils of healthy controls.