There is accumulating evidence that following bacterial infection, the massive recruitment and activation of the phagocytes, neutrophils, is accompanied with the extracellular release of active neutrophil elastase (NE), a potent serine protease. Using NE-deficient mice in a clinically relevant model of Pseudomonas aeruginosa-induced pneumonia, we provide compelling in vivo evidence that the absence of NE was associated with decreased protein and transcript levels of the proinflammatory cytokines TNF-α, MIP-2, and IL-6 in the lungs, coinciding with increased mortality of mutant mice to infection. The implication of NE in the induction of cytokine expression involved at least in part Toll-like receptor 4 (TLR-4). These findings were further confirmed following exposure of cultured macrophages to purified NE. Together, our data suggest strongly for the first time that NE not only plays a direct antibacterial role as it has been previously reported, but released active enzyme can also modulate cytokine expression, which contributes to host protection against P. aeruginosa. In light of our findings, the long held view that considers NE as a prime suspect in P. aeruginosa-associated diseases will need to be carefully reassessed. Also, therapeutic strategies aiming at NE inhibition should take into account the physiologic roles of the enzyme.