We used the Transwell system to select highly invasive cell lines from minimally invasive parent cells, and we compared gene expression in paired cell lines with high and low invasive potentials. AxI was relatively overexpressed in the highly invasive cell lines when compared with their minimally invasive counterparts. However, there is only limited information about the role of AxI in cancer invasion. The biologic function of AxI in tumor invasion was investigated by overexpression of full-length AxI in minimally invasive cells and by siRNA knockdown of AxI expression in highly invasive cells. Overexpression of AxI in minimally invasive cells increased their invasiveness. siRNA reduced cell invasiveness as AxI was downregulated in highly invasive cells. We further investigated the protein expression of AxI by immunohistochemistry and its correlation with clinicopathologic features. Data from a study of 58 patient specimens showed that AxI immunoreactivity was statistically significant with respect to lymph node status (P < .0001) and the patient's clinical stage (P < .0001). Our results demonstrate that AxI protein kinase seems to play an important role in the invasion and progression of lung cancer.