Neutrophils activated by bacteria release cytotoxic neutrophil extracellular traps (NETs) that can kill airway epithelial cells. However, molecular mechanisms that mitigate such damage are not clearly understood. Bacteria or bacterial components such as lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), a diacylglycerol mimetic, activate neutrophils to generate superoxide via NADPH oxidase (NOX) 2. Superoxide is eventually converted to hypochlorous acid (HOCl) by myeloperoxidase (MPO), subsequently to other reactive oxygen species (ROS), and induces NOX-dependent NET formation. Single pulmonary neuroendocrine cells (PNECs) or PNEC clusters (neuroepithelial bodies) present in airways secrete serotonin (5-hydroxytryptamine or 5-HT), a direct inhibitor and a competitive substrate of MPO. We hypothesized that serotonin secreted by PNECs into the lumen inhibits NET formation and protects epithelial cells against NET-mediated cell death. To test this hypothesis, neutrophils isolated from the peripheral blood of healthy human donors were stimulated to undergo NETosis, in the presence or absence of serotonin (Sytox Green assay; immunoconfocal microscopy). Both exogenous serotonin and 5-HT released by neuroendocrine H727 cells suppressed NOX-dependent ROS production and NETosis induced by PMA and lipopolysaccharide (In-cell Enzyme-linked immunosorbant assay; dihydrorhodamine DHR123 assay). By contrast, serotonin did not suppress NOX-independent NETosis induced by calcium ionophores A23187 and ionomycin. Cell death and detachment assays showed that airway epithelial A549 cells are highly susceptible for NET-mediated killing (immunoconfocal microscopy). Therefore, we conclude that serotonin inhibits NOX-dependent NETosis by suppressing ROS production and protects epithelial cells from NET-mediated damage.