Objective This study was designed to determine whether CD133 can be a stem cell marker to identify stem cell populations from small cell lung cancer cell line H446. Methods CD133+ and CD133- sub-populations were sorted from H446 by magnetic cell separation (MACS) and characterized for their in vitro stem cell-like properties. Results CD133+ cells and CD133- cells displayed similar abilities of colony formation, self-renewal, proliferation, differentiation and invasion, as well as resistance to chemotherapy drugs. Furthermore, colony formation assays showed that more than 40% of the cells in both CD133+ and CD133- sub-populations could form large colonies capable of regenerating the unsorted population and forming tumors in nude mice. Conclusions These results suggest that CD133 alone cannot be used as a stem cell marker for small cell lung cancer cell line H446, and both CD133+ and CD133- sub-populations contain similar amounts of cancer stem cells.