Gliomas are characterized by excessive proliferation, diffuse infiltration and immunosuppression. Recent studies implicate a key role for a restricted population of glioma stem cells (GSCs) in glioma invasive growth and recurrence. Transforming growth factor (TGF)-β2 is a mediator of immunosuppression associated with malignant glioma and also influences pro-invasive functions. Temozolomide (TMZ), is a new alkylating agent with promising antitumour efficacy for malignant gliomas, and the effect of TMZ on GSCs invasion has not been known. To address this issue, we developed studies aimed at neurospheres from primary cultured glioma cells, due to the fact that since neurospheres can be enriched in GSCs, we could examine whether TMZ inhibits the invasion of GSCs. TMZ reduced the TGF-β2-mediated invasion, and down-regulated TGF-β2 expression at the mRNA and protein levels. Thus, these results indicate that TMZ, as a chemotherapeutic agent, can reduce the invasion of GSCs and their immunosuppressive activity. TMZ may be used as an immunomodulating agent for glioma therapy.