Introduction: Pseudomonas aeruginosa is the major respiratory pathogen in patients with cystic fibrosis (CF). P. aeruginosa secreted proteases, in addition to host proteases, degrade lung tissue and interfere with immune processes. In this study, we aimed at evaluating the possible anti-inflammatory effects of protease inhibitors Marimastat and Ilomastat in the treatment of P. aeruginosa infection. Methods: Lung infection with P. aeruginosa PAO1 strain was established in wild-type and CFTR-knockout C57BL/6 mice expressing a luciferase gene under control of bovine IL-8 promoter. After intratracheal instillation with 150µM Marimastat and Ilomastat, lung inflammation was monitored by in vivo bioluminescence imaging and bacterial load in the lungs was assessed. In vitro, effects of protease inhibitors on PAO1 growth and viability were evaluated. Results: Acute lung infection was established in both wild-type and CFTR-knockout mice. After 24 hours, the infection induced IL-8-dependent bioluminescence emission indicating lung inflammation. In infected mice with ongoing inflammation, intratracheal treatment with 150µM Marimastat and Ilomastat reduced the bioluminescence signal in comparison to untreated, infected animals. Bacterial load in the lungs was not affected by the treatment and in vitro the same dose of Marimastat and Ilomastat did not affect PAO1 growth and viability, confirming that these molecules have no additional anti-bacterial activity. Conclusions: Our results show that inhibition of protease activity elicits anti-inflammatory effects in CF mice with acute P. aeruginosa lung infection. Thus, Marimastat and Ilomastat represent candidate molecules for the treatment of CF patients, encouraging further studies on protease inhibitors and their application in inflammatory diseases.