Neutrophils apply several antimicrobial strategies including degranulation, phagocytosis, the generation of reactive oxygen species (ROS), and the release of neutrophil extracellular traps (NETs) to fight pathogens. Iron is considered to be an invaluable constituent of host immune defense and it plays a dual role in immunity. It is a well-known component of antimicrobial proteins and is a necessary microelement for pathogen survival. The aim of this study was to broaden the knowledge regarding the impact of iron on the function of neutrophils. Neutrophils from healthy blood donors, patients suffering from mild iron deficiency anemia and HL-60 cells differentiated toward granulocyte-like cells were incubated with Fe2+, Fe3+, or holo-transferrin (holo-Tf). Moreover, we isolated murine neutrophils of HFE gene knockout (KO) mice and mice fed iron deficient, iron equivalent and high-iron diets. We analyzed the release of NETs, phagocytosis, degranulation of azurophilic granules, ROS release, bactericidal activity of granulocytes against E. coli, and neutrophil elastase (NE) activity. We show that holo-Tf inhibits the release of NETs release stimulated by phorbol 12-myristate 13-acetate by inhibiting NE activity. Studies performed in mice models reveal that iron overload inhibits the release of NETs and ROS production in neutrophils isolated from HFE KO and mice fed a high-iron diet. No impact of a low-iron diet on neutrophil phagocytosis, ROS production, or NETs release was observed. Our study underscores the physiological significance of iron in neutrophil function, specifically in the release of NETs.