Glioblastoma are highly aggressive brain tumors with poor prognosis. While various dysregulation of signaling pathways in gliomas have been described, the identification of biomarkers and therapy targets remains an important task for novel diagnostic and therapeutic approaches. Here we described that the Suppressor of fused (also known as Sufu) is significantly down-regulated in high-grade gliomas, correlating with a poor prognosis. We demonstrated that ectopic expression of Sufu inhibited cell proliferation, invasion and vasculogenic mimicry. In addition, overexpression of Sufu reduced Gli reporter gene transcription activity and prevented Gli1 nuclear accumulation, whereas knockdown of Sufu reversed these effects. Furthermore, overexpressed Sufu sensitized glioblastoma to Temozolomide and Cyclopamine. Thus, Sufu is potential tumor suppressor and therapeutic target in glioblastoma.