Various malignant cancers have been found to contain a sub-population of stem cell-like tumour cells, or cancer stem cells (CSCs), however, culture methods for CSCs and the size of the fraction of CSCs in C6, which is a commonly used glioma cell line, remain controversial. In this study, we demonstrated that the C6 cell line contains a fraction of tumour cells that can form tumour spheres in a simplified serum-free neural stem cell medium and express CD133 and nestin, which are widely-used markers for brain CSCs. Immunohistochemistry and immunofluorescence confirmed the existence of CSCs both in the C6 cell line and C6 xenografts. Flow cytometry demonstrated that 4.02% of cells in the C6 cell line and 4.21% in the C6 xenografts presented as CSCs. These results confirm the fraction of CSCs in the C6 cell line and provide a simple and effective method for isolation of CSCs to study the initiation and progression of human glioma and, possibly, other malignant tumours.