Glioma is the most common malignant tumors in adult brains, and Notch signaling pathway plays an important role in cell differentiation. The aim of the present study is to investigate the role of Notch1 in the progression of glioma cancers and clarify the mechanism of Notch1 silencing on inhibiting the proliferation of glioma cancer cells. First, endogenous Notch1 expression was interfered with a lentiviral vector of Notch1 shRNA. RT-PCR and western blotting were used for detecting the expression of Notch1 mRNA and protein, respectively. MTT assay results demonstrated that transfection with Notch1 shRNA and treatment with MRK003, a Notch1 inhibitor, both inhibited the proliferation of glioma cancer cells (p < 0.01). The lentiviral vector of Notch1 shRNA transfected into U251 cells induced cell cycle arrest at G0/G1 phase by FACS with PI staining analysis. Meanwhile, the expression levels of LC3-II and Beclin1 significantly increase in Notch1 shRNA-transfected U251 cells, suggesting that cell autophagy was induced when interfering with Notch1 in glioma cells. The downstream transcription factors were also detected by RT-PCR and western blotting analysis, and the data showed that interference with Notch1 increased the expression level of Hes-1, but not Hes-5. Taken together, all the data obviously revealed that Notch1 played an important role in the progression of glioma cancers. The clarification of the mechanism will be helpful for the diagnosis of glioma cancer and would provide new clues to molecular targets for cancer therapy.