Glioblastoma is one of the most radioresistant tumors. Exposure of cells to ionizing radiation leads to formation of reactive oxygen species (ROS) that are associated with radiation-induced cytotoxicity. ROS scavengers, therefore, are one of the important factors in protecting cells against ROS injury during ionizing radiation exposure. In the present study, we isolated and established a radioresistant variant clone (RRC) from U251 human glioblastoma cell line and investigated the potential role of antioxidant enzymes in radioresistance of the glioblastoma cell line. RRC showed a higher radioresistance than the parent cell line as measured by clonogenic survival assay and showed delayed G2/M arrest. Antioxidant enzymes, such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), glutathione reductase (GR), were activated up to 5-fold in RRC compared to the parent cells after radiation. In addition, RRC also had cross-resistance to the antitumor agent cisplatin. Therefore, radioresistance and cross-resistance to chemotherapeutic agent in RRC might be due to the highly coordinated activation of antioxidant enzymes rather than a single enzyme alone.