Objective To explore the methods of isolation,culture and identification of brain tumor stem cells （BTSC）,and to study the difference of chemosensitivity between the BTSC and the glioma cells. Methods Three kinds of glioma cells were respectly seeded into serum-free medium and then disassociated each BTSC, the 形态 can be observed under the scanning electron microscope. The expression of CD133/GFAP of BTSC was detected by RT-PCR, the CCK8 was used to evaluated the difference of proliferation between the glioma cells and BTSC both of which are used after CDDP and VCR. Results The brain stem cells isolated from glioma, have the ability to propagate and differationate in vitro.The character of expression CD133 was proved by flow cytometry. Conclusion BTSC is existed in glioma, and BTSC is more resistant to chemical therapy than glioma cell.