Erythropoietin-Producing human Hepatocellular carcinoma (Eph) receptors are largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although Eph receptors and their ligand Ephrins role are well studied in developmental processes, their function in tobacco smoke (TS) induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly up regulated EphA2, EphrinA1 expression, disrupted the actin filaments, decreased E-Cadherin expression, and increased protein permeability whereas the focal adhesion protein Paxillin was un-affected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS induced hyper-permeability in BAEpCs. In addition, when BAEpCs monolayer transfected with EphA2 expressing plasmid and treated with recombinant EphrinA1, the trans-epithelial electrical resistance decreased significantly. Furthermore, TS down regulated E-Cadherin expression and induced hyper-permeability across BAEpC monolayer in Erk1/Erk2, p38 and JNK MAPK dependent manner. TS induced hyper-permeability in BAEpCs monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were un-affected. The present data suggests that TS causes a significant damage to the BAEpCs via induction of EphA2 and down regulation of E-Cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.