Oxidative stress is a key mechanism underlying ozone-induced lung injury. Mitochondria can release mitochondrial reactive oxidative species (mtROS), which may lead to the activation of NLRP3 inflammasome. The goal of this study was to examine the roles of mtROS and NLRP3 inflammasome in acute ozone-induced airway inflammation and bronchial hyperresponsiveness (BHR). C57/BL6 mice (n = 8/group) were intraperitoneally treated with vehicle (phosphate buffered saline, PBS) or Mito-TEMPO (mtROS inhibitor, 20 mg/kg), or orally treated with VX-765 (caspse-1 inhibitor, 100 mg/kg) 1 hour before the ozone exposure (2.5 ppm, 3 hours). Compared to PBS-treated ozone-exposed mice, mitoTEMPO reduced the level of total malondialdehyde in bronchoalveolar lavage (BAL) fluid and increased the expression of mitochondrial complexes II and IV in the lung 24-hour after single ozone exposure. VX-765 inhibited ozone-induced BHR, BAL total cells including neutrophils and eosinophils and BAL inflammatory cytokines including IL-1α, IL-1β, KC and IL-6. Both mitoTEMPO and VX-765 reduced ozone-induced mtROS and inhibited capase-1 activity in lung tissue whilst VX-765 further inhibited DRP1 and MFF expression, increased MFN2 expression and down-regulated caspase-1 expression in lung tissue. These results indicate that acute ozone exposure induces mitochondrial dysfunction and NLRP3 inflammasome activation, while the latter has a critical role in the pathogenesis of ozone induced airway inflammation and BHR.