It is known that ozone stress can induce airway hyperresponsiveness (AHR). The underlying cellular and molecular mechanisms are not fully understood. We constructed a successive ozone-stressed rat model and showed that AHR caused by ozone stress presented as increased lung resistance (R(L)) to inhaled histamine but not baseline R(L). Meanwhile, structural disruption and decreased expression of integrin beta4 on airway epithelia were observed. Further regression analysis revealed a significant negative correlation between increases in R(L) to histamine (at 0.32 mg/ml) and mRNA expression of integrin beta4. Moreover, when integrin beta4 on human bronchial epithelial cells was knocked down, we found that reactive oxygen species was increased and apoptosis rates were higher. Overall, this study suggests that downregulation of integrin beta4 is important for the development ozone stress-induced AHR, presumably because it causes increased oxidative damage and epithelial apoptosis.