Background: Tumor necrosis factor-alpha (TNF?) disrupts the alveolar barriers in lung injuries due to various causes, for which glucocorticoids(GCs) are often used for the treatment. However, the involvement of GCs in the restoration of alveolar epithelial(AE) barrier dysfunction remains unclear. Aims and Objectives: To identify the effect of dexamethasone (Dex) on TNF?-induced AE barrier dysfunction. Methods: Rat type II AE cells isolated from 5?6-week old male Sprague Dawley rats were seeded onto collagen-coated transwells and transdifferentiated into type I-like cells to make confluent monolayer. It was stimulated with TNF? and/or Dex. Barrier function and cytotoxicity were evaluated by trans-epithelial electrical resistance (TER) and LDH assay, respectively. The expressions of tight junction(TJ)-related proteins were analyzed by qPCR, immunoblot and immunocytochemistry. Results: TNF? decreased TER, increased LDH release, and reduced the levels of a TJ protein, zonula occledens-1 (ZO-1), at 48 h of exposure (p<0.05). Dex added at 24 h of TNF? exposure restored the decline in TER at 48 h (p<0.01), which was suppressed in the presence of RU486, a glucocorticoid receptor (GR) antagonist (p<0.05). Dex did not reduce TNF?-induced cytotoxicity or ZO-1 protein levels, but augmented the ZO-1 immunoreactivity at intercellular junction sites. Dex significantly reduced the levels of myosin light chain kinase (MLCK) mRNA and phosphorylated myosin light chain (pMLC) protein, which can regulate ZO-1 distribution and stabilization at TJs. Conclusion: Dex resorted TNF?-induced AE barrier dysfunction by redistributing ZO-1 into intercellular junction sites probably via GR/MLCK/pMLC axis.