Nebulization of hypoxic human umbilical cord mesenchymal stem cell-derived extracellular vesicles (Hypo-EVs) can suppress airway inflammation and remodeling in a chronic asthmatic mouse; however, the exact mechanism remains unclear. Recently, airway epithelial barrier defects have been regarded as crucial therapeutic targets in asthma. The aim of this study was to investigate whether and how Hypo-EVs protect against the disruption of the airway epithelial barrier under asthmatic conditions.