Neutrophils release neutrophil extracellular traps (NETs) to trap and kill pathogens. The molecular mechanism linking reactive oxygen species (ROS) production to chromatin decondensation and antimicrobial protein binding is unknown. This study shows that upon activation, neutrophil elastase (NE) escapes from azurophilic granules and translocates to the nucleus, where it partially degrades histones, promoting chromatin decondensation. Myeloperoxidase (MPO) synergizes with NE in driving chromatin decondensation, independent of its enzymatic activity. NE knockout mice do not form NETs in a pulmonary model of Klebsiella pneumoniae infection, suggesting that this defect contributes to their immune deficiency. This work reveals a novel function for serine proteases and highly charged granular proteins in regulating chromatin density and provides evidence for a novel pathway allowing granular proteins to leak into the cytoplasm during oxidative burst.Neutrophils release neutrophil extracellular traps (NETs) to trap and kill pathogens. The molecular mechanism linking reactive oxygen species (ROS) production to chromatin decondensation and antimicrobial protein binding is unknown. This study shows that upon activation, neutrophil elastase (NE) escapes from azurophilic granules and translocates to the nucleus, where it partially degrades histones, promoting chromatin decondensation. Myeloperoxidase (MPO) synergizes with NE in driving chromatin decondensation, independent of its enzymatic activity. NE knockout mice do not form NETs in a pulmonary model of Klebsiella pneumoniae infection, suggesting that this defect contributes to their immune deficiency. This work reveals a novel function for serine proteases and highly charged granular proteins in regulating chromatin density and provides evidence for a novel pathway allowing granular proteins to leak into the cytoplasm during oxidative burst.