Abstract Ambient ozone has been linked to the worsening of symptoms of patients with obstructive diseases such as chronic obstructive pulmonary disease (COPD) and asthma. We investigated the role of cathepsin S on ozone-induced airway hyperresponsiveness (AHR) and inflammation, using the selective cathepsin S inhibitor, Compound A. Balb/c mice were exposed to ozone at a concentration of 3 ppm or air for 3 h, following administration by gavage of Compound A or vehicle. Bronchoalveolar lavage (BAL) was performed 3 h and 20–24 h following exposure, AHR was measured at 20–24 h only. Ozone exposure, compared to air exposure increased BAL cathepsin S levels, AHR and BAL inflammatory cells. Compound A (30 mg kg −1 p.o.) dosing compared to vehicle dosing inhibited ozone-induced AHR (−log PC 100 vehicle: −0.70 ± 0.12, n = 8 vs. cathepsin S inhibitor: −1.30 ± 0.06, P < 0.001, n = 8) at 20–24 h and BAL neutrophilia at 3 h and 20–24 h ( P < 0.05, n = 6). Ozone exposure increased levels of BAL cytokines IL-6, TNF-α and IFN-γ. Compound A reduced IL-6 at 3 h and 20–24 h ( P < 0.05, n = 5) and TNF-α, at 20–24 h ( P < 0.05, n = 6). These data indicate an important role for cathepsin S in the regulation of ozone-induced AHR and neutrophil cell recruitment and suggest that cathepsin S may be a target in the treatment of oxidative stress-induced AHR and inflammation.