Neutrophil serine proteases (NSPs), especially elastase, are major agents of lung destruction in cystic fibrosis (CF) patients. This study investigated SerpinB1, a highly efficient inhibitor of NSPs, in CF lung disease. Bronchoalveolar lavage fluid (BALF) from 31 children with CF and 24 control children was examined for amount and molecular species of SerpinB1, and its mechanism of action was studied. CF BALF had more SerpinB1 than control BALF (geometric mean 3.9 (95% CI 2.60-5.62) versus 1.37 (1.20-1.55) μg·mL⁻¹; p<0.001). BALF levels of SerpinB1 were higher for infected versus uninfected CF subjects (5.5 versus 2.7 μg·mL⁻¹; p<0.04) and substantially higher for elastase-positive versus -negative CF subjects (8.41 versus 1.89 μg·mL⁻¹; p<0.001). Most SerpinB1 in CF BALF had been cleaved. Adding recombinant SerpinB1 to CF BALF stoichiometrically inhibited endogenous elastase, indicating that the inhibitor functions in the CF microenvironment. In vitro simulations comparing SerpinB1 and α₁-antitrypsin (SerpinA1) showed that both rapidly form irreversible inhibitory covalent complexes with elastase and that these differed in survival time. The SerpinB1-elastase complex survived only briefly due to fragmentation of bound elastase, releasing cleaved SerpinB1, the molecular form in CF BALF. The findings define an innate role for SerpinB1 in CF airways.