Although chronic inhalation of endotoxin or lipopolysaccharide (LPS) causes all of the classic features of asthma, including airway hyperreactivity, airway inflammation, and airway remodeling, the mechanisms involved in this process are not clearly understood. The objective of this study was to determine whether intratracheal treatment with lipopolysaccharide antagonist (E5564, a lipid A analog) prevented the development of chronic endotoxin-induced airway disease in a mouse model of environmental airway disease. Pretreatment with 10 and 100 µg of E5564 was found to inhibit the airway response (hyperreactivity and inflammation) for up to 48 hours after the administration of the compound. Repeated dosing with 50 µg of E5564 intratracheally did not cause any measurable toxicity. Therefore, in a chronic experiment, mice were treated with either E5564 (50 µg) or vehicle 3 times weekly for 5 weeks and simultaneously daily exposed to either LPS (4.65 ± 0.30 µg/m³) or saline aerosol. E5564 was effective in decreasing the airway hyperreactivity to methacholine, the airspace neutrophilia, the interleukin-6 in the lung lavage fluid, and the neutrophil infiltration of the airways 36 hours after five weeks of LPS inhalation. Less collagen deposition was observed in the airways of E5564-treated mice compared to vehicle-treated mice after a four-week recovery period. Our results indicate that E5564, a toll-like receptor 4 antagonist, minimizes the physiologic and biologic effects of chronic LPS inhalation, suggesting a therapeutic role for competitive LPS antagonists in preventing or reducing endotoxin-induced environmental airway disease.