Klotho mice, which exhibit multiple phenotypes resembling human aging, develop pulmonary emphysema. In this study, to clarify the mechanism of their emphysematous change through development, we evaluated the expression of matrix metalloproteinase (MMP)-2, 9 and the tissue inhibitors of matrix metalloproteinase (TIMP)-1, 2 in the lungs of Klotho mice and wild type mice. Klotho mice showed obvious air space enlargement at 5 weeks of age, but not at 2 weeks of age. Immunohistochemical analysis revealed that expression of MMP-9 was increased in Klotho mice compared with wild type at 5 weeks of age. Western blot analysis and gelatin zymography also revealed that the expression and the gelatinolytic activity of MMP-9 were increased in the lungs of Klotho mice. The expression of TIMP-1 decreased in the lungs of Klotho mice. MMP-2 and TIMP-2 showed no significant differences at 5 weeks of age. At 2 weeks of age, there were no significant differences in the expressions of MMP-9 and TIMP-1 between Klotho and wild type mice. These findings suggest that imbalance of MMP-9 and TIMP-1 is associated with the development of pulmonary emphysema in Klotho mice.