Abnormality in mitochondria has been suggested to be associated with development of allergic airway disorders. In this study, to evaluate the relationship between mitochondrial reactive oxygen species (ROS) and NLRP3 inflammasome activation in allergic asthma, we used a newly developed mitochondrial ROS inhibitor, NecroX-5. NecroX-5 reduced the increase of mitochondrial ROS generation in airway inflammatory cells, as well as bronchial epithelial cells, NLRP3 inflammasome activation, the nuclear translocation of nuclear factor-κB, increased expression of various inflammatory mediators and pathophysiological features of allergic asthma in mice. Finally, blockade of IL-1β substantially reduced airway inflammation and hyperresponsiveness in the asthmatic mice. These findings suggest that mitochondrial ROS have a critical role in the pathogenesis of allergic airway inflammation through the modulation of NLRP3 inflammasome activation, providing a novel role of airway epithelial cells expressing NLRP3 inflammasome as an immune responder.