Abstract: Objective To investigate the role of epidermal growth factor receptor (EGFR) signaling pathway in bronchial epithelial actin stress fiber (F-actin) rearrangement induced by house dust mite (HDM). Methods Normal human bronchial epithelial cells (16HBE) were stimulated with HDM with or without pretreatment with AG-1478, an EGFR inhibitor. The levels of phospho(p)-EGFR, F-actin, E-cadherin and β-catenin in the cell cultures were detected with Western blotting. The localizations of F-actin, E-cadherin and β-catenin in the bronchial epithelial cells were determined with immunofluorescence assay, and the transmembrane electrical resistance (TER) and FITC-dextran flux (FITC-DX) in the cells were measured to assess the barrier function of the bronchial epithelia. Results HDM stimulation of the cells for 10 min resulted in significantly increased p-EGFR expression (P<0.05) without causing obvious changes in the expression of E-cadherin (P>0.05) or β-catenin (P>0.05). Immunofluorescence assay revealed delocalization of E-cadherin and β-catenin in HDM-treated 16HBE cells, shown by their diffusion from the cell membrane to the cytoplasm. In HDM-treated cells, the TER was significantly decreased to (70.00±4.33)% and the FITC-DX was significantly increased to (115.98±4.34)%; Inhibition of EGFR reversed the delocalization of E-cadherin and β-catenin, improved the TER to (90.00 ± 3.75)% and lowered the FITC-DX to (101.10 ± 2.10)%. HDM induced increased expression and rearrangement of F-actin, which was obviously inhibited by pretreatment of the cells with AG-1478 (P<0.05). Conclusion EGFR signaling pathway mediates HDM-induced F-actin rearrangement in human bronchial epithelial cells to contribute to epithelial barrier dysfunction.