Mycoplasma pneumoniae (MP) may be associated with chronic asthma. Our previous studies demonstrate that airway MP infection in mice can cause bronchial hyperresponsiveness (BHR) and airway inflammation. Inhaled corticosteroids are the mainstay for asthma treatment, but their effects on BHR and airway inflammation associated with MP are uncertain. In this study, we hypothesized that inhaled steroids could reduce or eliminate lung MP infection and improve BHR and airway inflammation. To test our hypothesis, two groups of mice were studied. The first group received an aerosolized fluticasone at 100 μg/mL for 5 days with MP infection at day 3. Three days (day 6) after the infection, the mice were tested for MP, airway inflammation, and lung resistance (RL) to aerosolized methacholine challenges (1.6 to 100 mg/mL). The control mice were similarly treated with an aerosolized sham solution and infected with MP. MP culture and quantitative real-time polymerase chain reaction (PCR) were performed on both BAL samples and lung tissues. By culture, MP in the lung tissue but not in the BAL was significantly reduced in the mice treated with fluticasone compared with the control mice (geometric mean, 4.7 × 105 vs 100 × 105 color-changing units, respectively, p < 0.05). This was confirmed by real-time PCR, which demonstrated a threefold decrease of MP in the lung tissue of mice with fluticasone. Interestingly, within the group of mice with fluticasone, the amount of MP in the BAL was 2.5-fold to sixfold higher than in the lung tissue (p = 0.06). This was opposite of findings in the control mice, which had more MP (threefold to fourfold) in the lung than in the BAL. RL was significantly reduced in the mice with fluticasone at methacholine doses of 1.6, 50, and 100 mg/mL compared with the control mice. Consistent with RL, BAL neutrophils and lung tissue inflammation were also significantly decreased in the mice with fluticasone. The amount of MP in the lung tissue was correlated with RL at methacholine, 1.6 mg/mL (culture, ρ = 0.44, p = 0.066), 25 mg/mL (culture, ρ = 0.50, p = 0.03), and 100 mg/mL (culture, ρ = 0.55, p = 0.01; PCR, ρ = 0.41, p = 0.09). Our data suggest that fluticasone blocks the adherence of MP in the lung tissue, leading to the increased MP in the airway lumen. BHR and airway inflammation were also reduced by fluticasone treatment, which might result from both antimicrobial and direct anti-inflammatory effects of fluticasone.