The airway epithelium lining the luminal surface of the respiratory tract creates a protective barrier that ensures maintenance of tissue homeostasis and prevention of respiratory diseases. The airway epithelium, unfortunately, is frequently injured by inhaled toxic materials, trauma, or medical procedures. Substantial or repeated airway epithelial injury can lead to dysregulated intrinsic repair pathways and aberrant tissue remodeling that can lead to dysfunctional airway epithelium. While disruption in the epithelial integrity is directly linked to degraded epithelial barrier function, the correlation between the structure and function of the airway epithelium remains elusive. In this study, we quantified the impact of acutely induced airway epithelium injury on disruption of the epithelial barrier functions. By monitoring alternation of the flow motions and tissue bioimpedance at local injury site, degradation of the epithelial functions, including mucociliary clearance and tight/adherens junction formation, were accurately determined with a high spatiotemporal resolution. Computational models that can simulate and predict the disruption of the mucociliary flow and airway tissue bioimpedance have been generated to assist interpretation of the experimental results. Collectively, findings of this study advance our knowledge of the structure–function relationships of the airway epithelium that can promote development of efficient and accurate diagnosis of airway tissue injury.