To explore the immunogenicity of glioma stem-like cell-associated antigens (SAAs) from sorted or unsorted glioma tumor stem-like cells (TSCs) as well as irradiated TSCs. Two primary human malignant glioma lines (SHG62, SHG66) and U87 cell line were primarily cultured in the serum-free medium (SFM) supplemented with EGF/bFGF. TSCs were identified by their self-renewal, multi-lineage differentiation and tumorigenic activity. To prepare SAAs in vitro, CD133+ TSCs were sorted either by magnetic cell sorting or with irradiation (6 Gy).The cytotoxicity induced by autogenous myeloid dendritic cell (DC)-mediated SAA-specific cytotoxic T lymphocytes (CTLs) was assessed by the Just Another Method test. SHG62, SHG66, and U87 cells contained TSCs. CD133+ SAAs-specific CTLs were significantly more cytotoxic than effector cells loaded with unsorted SAA (P < 0.05). Effector cells loaded with irradiated SAAs were more cytotoxic than those with regular SAAs (P < 0.01). SAAs from CD133+ TSCs and irradiated TSCs provide highly immunogenic antigens. TSCs might be a novel source of antigens for DC vaccination against malignant gliomas.