Background and Aim: Recently, it has been recognized that asthma and COPD can coexist as asthma-COPD overlap (ACO). Due to the difference of clinical course, it is thought the pathogenesis of ACO is different from that of asthma or COPD; however, its animal models have not yet been established. In animal experiments, papain (a cysteine protease enzyme present in papaya) is known to cause asthmatic response by daily inhalation, and lung emphysema by intermittent inhalation. Here we developed ACO mice model by weekly intratracheal administrations of papain, and we examined whether the mice model well reflects the features of ACO, comparing its features with elastase induced COPD model. Methods: PBS, papain, or porcine pancreatic elastase (PPE) was intratracheally administered to female C57BL/6N mice (5-6 weeks old) on days 0, 7, 14, 21, while PPE on day 0 only. The mice were sacrificed on day 25. Lung mechanics, airway responsiveness to methacholine, bronchoalveolar lavage fluid (BALF), lung tissue, and mRNA in lung homogenates were analyzed. Results: In lung mechanics tests, both papain and PPE group showed significantly higher compliance and inspiratory capacity than PBS group, while the airway responsiveness to methacholine was significantly higher in papain group than in other groups. Papain increased eosinophils in BALF. Both papain and PPE induced alveolar air-space enlargement. In addition, type 2 inflammatory cytokines such as IL-13 and IL-25 were significantly higher in papain group than in other groups. Conclusions: Weekly papain administrations induced ACO like features in mice. Clarification of the underlying pathogenesis and development of novel treatment might be expected using this single-agent simple model.