Dual oxidase (Duox) 1 and Duox2 are important sources of hydrogen peroxide production and play a role in host defense in airways. Little is known about their regulation in association with smoking or chronic obstructive pulmonary disease (COPD). We investigated the epithelial expression of Duox1 and Duox2 in the airways of smokers, and the relationship between this expression and COPD at early stage. First, using bronchoscopy, we harvested tracheal and bronchial epithelium from individuals who have never smoked and current smokers. Duox1 expression in brushed tracheal and bronchial epithelium was significantly downregulated, whereas Duox2 was upregulated, in current smokers as compared to individuals who have never smoked. Second, laser capture microdissection and microscope-assisted manual dissection were performed in surgically resected lung tissues to collect bronchiolar epithelium and alveolar septa. Subjects with mild/moderate COPD, who were all former smokers, exhibited downregulation of bronchiolar Duox1 and Duox2 when compared to individuals who have never smoked, whereas a difference between former smokers, with and without COPD, was observed only for Duox1. Alveolar Duox1 and Duox2 expression was low and did not differ among the groups. These results imply that the airway expression of Duox1 and Duox2 is diversely associated with smoking and COPD.