Background: Ciprofloxacin DPI is in Phase III development for chronic intermittent treatment of non-cystic fibrosis bronchiectasis (NCFB) patients. Objective: To assess the bactericidal activity of Ciprofloxacin DPI against clinical respiratory pathogens in a HF system. Methods: Recent respiratory isolates of ciprofloxacin-susceptible H. influenzae, K. pneumoniae, S. aureus (1 each), and 3 P. aeruginosa (minimum inhibitory concentrations [MICs] of 0.12, 0.5, and 1.0mg/L) were cultured in a HF system and exposed to ciprofloxacin at levels simulating the mean sputum concentration profile after patient inhalation of Ciprofloxacin DPI 32.5mg. Colony-forming units (CFUs) and MICs were measured during a 5-day bid treatment course and 5 days of untreated follow-up. Results: Rapid bactericidal killing of all organisms was observed within 10 hours of first treatment and bacterial populations were reduced below the limit of detection (10 CFU/mL) during treatment. In the follow-up phase, no regrowth was observed for H. influenzae or the P. aeruginosa isolate with MIC of 0.12mg/L. Remaining isolates showed regrowth without elevated MICs. There were no changes in resistance frequency at ciprofloxacin doses of 3X and 5X MIC compared with untreated isolates. Conclusion: In this in vitro infection model, Ciprofloxacin exhibited bactericidal activity against clinical respiratory pathogens at concentrations achieved in human sputum after inhalation of Ciprofloxacin DPI. Isolates that regrew did not show elevated MICs or increased resistance, suggesting that Ciprofloxacin DPI does not promote resistance development. Clinical data in NCFB patients are required to confirm results.