Proteasome dysfunction is emerging as a novel pathomechanism for the development of chronic obstructive pulmonary disease (COPD), a major leading cause of death in the world. Cigarette smoke, one of the main risk factors for COPD, impairs proteasome function in vitro and in vivo. In the present study, we dissected the molecular changes induced by cigarette smoke on the proteasome in lung epithelial cells and mouse lungs. 26S proteasome pull-down, MS interactome, and stoichiometry analyses indicated that 26S proteasome complexes become instable in cigarette smoke-treated lung epithelial cells as well as in lungs of mice after three day smoke exposure. The interactome of the 26S was clearly altered in mouse lungs upon smoke exposure but not in cells after 24 h of smoke exposure. Using native MS analysis of purified 20S proteasomes, we observed some destabilization of 20S complexes purified from cigarette smoke-exposed cells in the absence of any dominant and inhibitory modification of proteasomal proteins. Taken together, our results suggest that cigarette smoke induces minor but detectable changes in the stability of 20S and 26S proteasome complexes which might contribute to imbalanced proteostasis in a chronic setting as observed in chronic lung diseases associated with cigarette smoking.