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Glioma-Associated Oncogene Family Zinc Finger 2 (Gli2) seems to be the major nuclear effector of Sonic Hedgehog (SHH) signaling to regulate self-renewal and tumorigenic potential of Glioblastoma multiforme (GBM) cells. Three phosphorylated peptides derived from Gli2 were synthesized and combined with cell-penetrating peptide Tat-(47–57) (AYGRKKRRQRRR). Western Blot was applied to detect the phosphorylation level of Gli2 and cell division protein kinase 6 (CDK6) luciferase reporter was utilized to detect the transcriptional activator function of Gli2. Clonogenic survival assay and apoptosis assay were used to testify the radiosensitization effect. The mixed three phosphorylated peptides derived from Gli2 increased the phosphorylation level of Gli2 and decreased Gli2 transcriptional activator activity significantly than the individually used peptide. The mixed three phosphorylated peptides showed greater radiation-sensitizing effects in GBM cells in clonogenic and survival assay compared with control peptide. We present here a novel rational strategy for developing phosphorylated peptides derived from Gli2 to decrease Gli2 transcriptional activator activity and such administration could radiosensitize GBM.