Lung cancer is a leading cause of death in human. Cancer stem cells have been regarded as basis for failure of current therapeutic options. Salinomycin was shown to kill these cancer stem cells in some types of cancer such as breast cancer and leukemia. The in vitro anticancer activities of salinomycin have been validated against the lung cancer cell line A549 via sulforhodamine B and colony formation assay. Salinomycin has been demonstrated to significantly rupture the in vitro lung cancer tumorospheres from ALDH positive A549 lung cells using flow cytometry. Expression of stem cell markers OCT-4, NANOG and SOX2 in ALDH positive A549 lung cells was decreased significantly by real-time RT-PCR analysis after 24 hour salinomycin treatment. Taken together, salinomycin may provide a promising approach for lung cancer chemotherapy.