Tracheal reconstruction has been an important issue in clinic, but it is limited for the ability of epithelial regeneration. Several reports have shown that stromal cell-derived factor-1 (SDF-1) and chemokine receptor CXCR4 play an important role in cell proliferation and migration of multiple cell types. But there is no report of SDF-1 and CXCR4 in tracheal cells. In this paper, the rat tracheal epithelial cells covered with cilium were isolated and cultured using two enzyme digestions, and CXCR4 lentivirus was constructed and infected to the tracheal cells successfully. The results showed that the expression of CXCR4 which was covered on cellular membrane majorly was low in normal cells, and the cell proliferation was increased accompanied with the increase in SDF-1 concentration. The cell proliferation, migration and intracellular free calcium were increased significantly in CXCR4 lentivirus infected groups in a dose-dependent manner, and these effects could be inhibited after CXCR4 inhibitor AMD3100 treated because the expression of CXCR4 was decreased. Our findings indicate that the activation of CXCR4 may promote tracheal cell proliferation and migration to the sites of airway injury where SDF-1 is regulated.