This study investigates the role of histone hypercitrullination in chromatin decondensation and neutrophil extracellular trap (NET) formation. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. The authors show that PAD4-mediated histone hypercitrullination mediates chromatin decondensation in HL-60 granulocytes and blood neutrophils. Inhibition of PAD4 decreases histone hypercitrullination and NET-like structures, while PAD4 treatment facilitates these processes. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. PAD4 activation leads to the loss of heterochromatin and multilobular nuclear structures. Additionally, citrullination of avian nucleosome arrays inhibits their compaction by linker histone H5, suggesting that histone hypercitrullination plays a crucial role in chromatin decondensation during NET formation. These findings support a model where global histone hypercitrullination regulates chromatin unfolding during NET formation.This study investigates the role of histone hypercitrullination in chromatin decondensation and neutrophil extracellular trap (NET) formation. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. The authors show that PAD4-mediated histone hypercitrullination mediates chromatin decondensation in HL-60 granulocytes and blood neutrophils. Inhibition of PAD4 decreases histone hypercitrullination and NET-like structures, while PAD4 treatment facilitates these processes. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. PAD4 activation leads to the loss of heterochromatin and multilobular nuclear structures. Additionally, citrullination of avian nucleosome arrays inhibits their compaction by linker histone H5, suggesting that histone hypercitrullination plays a crucial role in chromatin decondensation during NET formation. These findings support a model where global histone hypercitrullination regulates chromatin unfolding during NET formation.