In order to explore the function of EphA2 gene on the proliferation, apoptosis, motility and invasion of glioma cell line U251. The EphA2 mRNA levels of normal brain and two glioma cell lines U251 and U87 were detected by reverse transcription PCR (RT-PCR). Then, the function of EphA2 in glioma cell line U251 was analyzed by RNAi technology. The expression level of EphA2 gene was higher in U251 and U87 cells than that in normal brain. siRNA targeting EphA2 was designed and synthesized and then the siRNA was transfected into U251 cell with a high transfection efficiency of 70.31%. 50 nmol/L of siRNA could lead to over 70.0% reduction in EphA2 protein and mRNA level detected by Western blot and RT-PCR respectively. The cell proliferation was notably attenuated and the apoptosis was significantly increased after EhpA2 was downregulated. The migration and invasion of glioma cells were also attenuated when EhpA2 was knocked down by siRNA. EphA2 plays an important role in the malignance of glioma U251 cells and may be a novel molecular marker for the malignance of the glioma cell line U251. EphA2 could be a potential target in gene therapy for glioma.