ABSTRACT Rationale: Chlorine gas (Cl2) inhalation causes oxidative stress, airway epithelial damage, airway hyperresponsiveness (AHR) and neutrophilia. Objectives: We evaluated the effect of neutrophil depletion on Cl2-induced AHR, affect the endogenous anti-oxidant response and, if eosinophils or macrophages influence Cl2-induced AHR. Methods: We exposed male Balb/C to 100ppm Cl2 for 5 minutes. We quantified inflammatory cell populations in bronchoalveolar lavage (BAL), and the anti-oxidant response in lung tissue by qPCR, as well as and nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation by immunofluorescence. In vitro, NRF2 nuclear translocation in response to exogenous HOCl was assessed using a luciferase assay. Anti-Gr1 antibody or anti Ly6G were used to deplete neutrophils. Effect of neutrophil depletion on IL-13 and IL-17 were measured by ELISA. Eosinophils and macrophages were depleted using TRFK5 or clodronate-loaded liposomes, respectively. AHR was evaluated with the constant phase model in response to inhaled aerosolized methacholine. Main results: Cl2 induced neutrophilia and increased expression of NRF2 mRNA, superoxide dismutase-1 and heme-oxygenase 1. Neutrophil depletion abolished Cl2-induced AHR in large, conducting airways and prevented increases in anti-oxidant gene expression and NRF2 nuclear translocation. Exogenous hypochlorite administration resulted in increased NRF2 nuclear translocation in vitro. Following Cl2, neutrophils occupied 22 +/-7% of the luminal space in large airways. IL-17 in BAL was increased following Cl2, an effect prevented by neutrophil depletion. Neither depletion of eosinophils or macrophages prevented Cl2-induced AHR. Conclusion: Neutrophils increase AHR by molecular and mechanical mechanisms by increasing oxidative stress and occupation of luminal space in large airways.