Glioma, the most common tumor of the central nervous system (CNS), currently results in a high rate of morbidity and mortality. The expression of CD133, a stem-like cell marker expressed in the glioma cells, is believed to lead to tumorigenesis in the human brain. Thus, it is necessary to find a proper method to specifically kill the CD133 + glioma cells. Dendritic cell (DC)/tumor hybrids are proven to be able to induce an effective immune response, leading to killing of glioma cells in vitro. We isolated CD133 + cells from a population of primary glioma cells, and cultured autologous DCs and T cells at the same time. Next, we electrofused the DCs with the CD133 + glioma cells and with CD133− ones, in order to explore a new strategy for glioma therapy. We then exposed the T cells to five separate groups of cells: DC/CD133 + hybrids, DC/CD133 − hybrids, DCs alone, unsorted glioma cells alone and mixed DCs-glioma cells. A cytotoxicity assay showed that T cells stimulated by either type of hybrid were able to kill cultured autologous glioma cells significantly more effectively than those stimulated by the other three cell types (P<0.05). The amounts of IFN-γ secreted by T cells stimulated by the two types of fused cells were obviously increased compared to those stimulated by the other three cell types (P<0.05). However, no significant differences were noted between the effects of the two hybrids, neither in the cytotoxicity assay nor in the IFN-γ release assay (P>0.05). Therefore, both DC/CD133 + and DC/CD133 − hybrids can cause significant T cell immune responses in vitro. There were no significant differences between the immune responses caused by the two types of hybrids.