Cancer stem cells (CSCs) are the sole population possessing high self-renewal activity in tumors, with their existence affecting tumor recurrence. However, the invasive activity of CSCs has yet to be fully understood. In this article, we established a tumor sphere culture of RSV-M mouse glioma cells (RSV-M-TS) and evaluated their migration and invasion activities. Histological analysis of a tumor formed by cranial injection of the RSV-M-TS cells showed highly invasive properties and similarities with human malignant glioma tissues. When the migration activity of both RSV-M and RSV-M-TS cells were compared by intracranial injection, rapid migration of RSV-M-TS cells was observed. To confirm the invasive capabilities of RSV-M-TS cells, a three-dimensional collagen invasion assay was performed in vitro using RSV-M, RSV-M-TS, and RSV-M-TS cells cultured with medium containing serum. RSV-M and RSV-M-TS cultured with medium containing serum for 8 days indicated low migration activity, while moderate invasion activity was observed in RSV-M-TS cells. This activity was further enhanced by incubation with medium containing serum overnight. To identify the genes involved in this invasion activity, we performed quantitative polymerase chain reaction (PCR) array analysis of RSV-M and RSV-M-TS cells. Of 84 cancer metastasis-related genes, up-regulation was observed in 24 genes, while 4 genes appeared to be down-regulated in RSV-M-TS cells. These results suggest that the enhanced invasive activity of glioma sphere cells correlates with a number of tumor metastasis-related genes and plays a role in the dissemination and invasion of glioma cells.