Cigarette smoking is a major factor for the development of pulmonary emphysema because it induces abnormal inflammation and a protease-rich local milieu that causes connective tissue breakdown of the lungs. As a result of its capacity to degrade lung tissue and the high risk of patients lacking α1-antitrypsin to develop emphysema, much interest has focused on neutrophil elastase (NE). Two similar neutrophil serine proteases (NSPs), cathepsin G and proteinase 3, coexist with NE in humans and mice, but their potential tissue-destructive role(s) remains unclear. Using a gene-targeting approach, we observed that in contrast to their wild-type littermates, mice deficient in all three NSPs were substantially protected against lung tissue destruction after long-term exposure to cigarette smoke. In exploring the underlying basis for disrupted wild-type lung air spaces, we found that active NSPs collectively caused more severe lung damage than did NE alone. Furthermore, NSP activities unleashed increased activity of the tissue-destructive proteases macrophage elastase (matrix metalloproteinase-12) and gelatinase B (matrix metalloproteinase-9). These in vivo data provide, for the first time, compelling evidence of the collateral involvement of cathepsin G, NE, and proteinase 3 in cigarette smoke-induced tissue damage and emphysema. They also reveal a complex positive feed-forward loop whereby these NSPs induce the destructive potential of other proteases, thereby generating a chronic and pathogenic protease-rich milieu.