Abstract: To investigate the biological functions of the ALDH1-positive cancer cells exhibiting the characteristics of a cancer stem cell in the A549 lung cancer cell line. Methods: Fluorescence-activated cell sorting  analysis was used to differentiate between the ALDH1-positive and ALDH1-negative cancer cells in an A549 lung cancer cell line. Tumor sphere cultivation, MTT, clonogenicity, and in vitro transwell assays as well as tumor initiation in vivo were used to compare the self-renewing, proliferative, clonogenic, and invasive abilities as well as the tumorigenicity between the ALDH1-positive, ALDH1-negative, and unsorted A549 cells. Results: The self-renewing, proliferative, clonogenic, and invasive abilities as well as the tumorigenicity of the ALDH1-positive cancer cells in the A549 lung cancer cell line were higher than those of the ALDH1-negative and unsorted A549 cells. Conclusion: The ALDH1-positive cancer cells in the A549 lung cancer cell line possess the characteristics of cancer stem cells and may be used as NSCLCSC-associated tumor markers.