Objective:To investigate the vasculogenic mimicry formation induced by hypoxia in Ⅱ-Ⅲ human glioma cell and the effect of alphastatin peptide suppressing the hypoxia-induced vasculogenic mimicry formation and the mechanism. Methods: MTT, Transwell and three- dimentional culture were used to detect the proliferation, migration and tubule formation of SHG44. (EphA2) and matrix metalloproteinases 2 (MMP2) was detected by RT-PCR and West blotting analysis. Results: The OD490 in hypoxia group was 0.60±0.06 and in control group was 0.46±0.05. The number of cell migration was 178.71±18.81 in hypoxia group and 85.86±17.92 in control group. The tubule formation was 56.80±12.21 in hypoxia group and 4.20±2.62 in control group. The proliferation, migration and tubule formation in hypoxia group were significantly higher than that in control group. The expression of VEGF-n, EphA2 and MMP2 was upregnlated in hypoxia. When various concentrations of alphastatin (100, 1 000, 10 000 nmol/L) were added to hypoxia group, the numbers of cell migration were 142.57±12.12, 92.71±17.68, 30.00±7.72 and the tubule formation were 47.71±10.58, 18.86±8.40, 8.43±5.62. The cell migration and tubule formation were significantly suppressed by alphastatin in a dose-dependent manner. In alphastatin group, the phosphorylation of EphA2 protein (P=0.037, F=4.629) and activation of MMP2 protein (P=0.005, F=9.331) were significantly suppressed but there was no MMP2 activation maybe the key pathway to form vasculogenic mimicry.