Objective To isolate and expand cancer stem cells in human lung cancer cell line NCI-H446 and to identify their biological properties. Methods NCI-H446 cells were cultured in serum-free conditions to generate tumor spheres. The tumor spheres were then expanded and cultured in a serum-containing medium to permit their differentiation.The proliferative capacity of the normal NCI-H446 cells and tumor sphere-forming cells were tested by MTT assay, and the tumorigenicity and invasion of the both normal and sphere-cells were evaluated using animal and Transwell invasion experiments.The expression of CD133 and CD44 on the surface of the normal NCI-H446 cells and tumor sphere-forming cells were detected by flow cytometry. Results A small number of floating tumor spheres were isolated and expanded in serum-free conditions and the cell of the spheres maintained a strong capacity for self-renewal and proliferation. These tumor spheres attached to the bottom of culture plates and began to differentiate in a serum-containing medium.The xenograft tumorigenicity of tumor sphere forming cells was significantly higher than that of the normal NCI-H446 cells while injecting 5×105 cells in animal experiments.The capability of invasion of tumor sphere forming cells was significantly higher than that of the normal NCI-H446 cells in transwell invasion experiments. The percentage of CD133,CD44 cell population was significantly higher in tumor spheres than that in the normal NCI-H446 cells. Conclusion Cancer stem cells exist in human lung cancer cell line NCI-H446 and can be isolated and expanded in serum