Immunostimulatory sequences of DNA (ISS) inhibit eosinophilic airway inflammation, Th2 responses, and airway hyperreactivity (AHR) in mouse models of acute ovalbumin (OVA)-induced airway inflammation. To determine whether ISS inhibits airway remodeling, we developed a mouse model of airway remodeling in which OVA-sensitized mice were repeatedly exposed to intranasal OVA administration for 1–6 mo. Mice chronically exposed to OVA developed sustained eosinophilic airway inflammation and sustained AHR to methacholine compared with control mice. In addition, the mice chronically exposed to OVA developed features of airway remodeling, including thickening of the peribronchial smooth muscle layer, peribronchial myofibroblast accumulation, expression of the profibrotic growth factor transforming growth factor-β, and subepithelial collagen deposition (assessed by quantitation of the area of peribronchial trichrome staining using image analysis, and immunostaining with anti–collagen V antibodies). Administration of ISS systemically every other week significantly inhibited the development of AHR, eosinophilic inflammation, airway mucus production, and importantly, airway remodeling in mice chronically exposed to OVA for 3–6 mo. In addition, ISS significantly reduced bronchoalveolar lavage and lung levels of the profibrotic cytokine transforming growth factor-β. These studies demonstrate that ISS prevents not only Th2-mediated airway inflammation in response to acute allergen challenge, but also airway remodeling associated with chronic allergen challenge.