Immunotherapy strategies targeting glioma stem-like cells (GSCs) hold promise for improving outcomes in glioblastoma patients. We used the A2B5 monoclonal antibody to classify GSCs derived from the mouse GL261 glioma cell line, and A2B5+ GL261 cell lysate-pulsed dendritic cells (DCs) were used to treat mouse glioma. We found that such DCs elicited a stronger specific cytotoxic T lymphocyte response against A2B5+ GL261 cells than A2B5- GL261 cell lysate-pulsed DCs. The effect of A2B5+ GL261 cell lysate-pulsed DCs in vivo depended on when the vaccination was started. In the tumor cell adaptation phase, C57BL/6 mice had an immune response against GL261, and vaccination enhanced the immune response and prevented glioma formation in 37.5% of mice. In contrast, after glioma formation, the immune response against GL261 was decreased, and vaccination had no therapeutic effect. Our findings suggest that vaccination with A2B5+ GL261 cell lysate-pulsed DCs only has some glioma preventive effect.