Relaxin is a reproductive hormone with pleiotropic actions. In addition to airway fibrosis, relaxin deficiency results in airway structural changes (epithelial thickening) and increased lung recoil, suggesting that relaxin may impact upon other aspects of airway/lung structure and function, beyond its ability to regulate collagen turnover. Furthermore, these structural changes associated with relaxin deficiency show marked similarity to the structural changes seen in asthma. The current study investigates the broader role of relaxin in regulating airway structure and function, and examines the relationship between airway inflammation, structural changes and airway hyperresponsiveness (AHR) using an ovalbumin (OVA)-induced model of allergic airways disease (AAD). The model of AAD was applied to 12-month old relaxin deficient (Rln-/-) mice with established airway fibrosis and to age-matched wild-type (Rln+/+) controls. OVA-treated Rln+/+ mice (induced inflammation) developed increased epithelial thickening (p<0.05) and AHR (p<0.05) but not airway fibrosis, compared to saline-treated Rln+/+ controls. Saline-treated Rln-/- mice had significantly increased lung collagen deposition (existing fibrosis) and epithelial thickening, and remarkably were found to have increased AHR that was equivalent to that in OVA-treated Rln+/+ mice (all p<0.05 vs saline-treated Rln+/+ controls). OVA-treated Rln-/- mice (existing fibrosis and induced inflammation) had increased airway/lung fibrosis (p<0.05), but equivalent airway inflammation and AHR compared to OVA-treated Rln+/+ animals. These findings demonstrate for the first time a role for relaxin in the regulation of airway responses using Rln-/- mice and suggest that airway fibrosis and/or epithelial thickening can result in increased AHR equivalent to that induced by airway inflammation in AAD.