Abstract: This essay was aimed to isolate, purify and characterize the protein with antifungal activity from arugula (Eruca sativa Mill.) seeds.The antifungal protein was isolated and purified from the seed of arugula by ion exchange chromatography on SP-Sepharose and followed by fast protein liquid chromatography on Mono S^TM 5/50 GL and Superdex^TM 75 GL.Tricine-SDS-PAGE was used to analysis its purity and molecular weight.The antifungal activity and stability were studied by means of paper diffusion and fluorescence staining.The results showed that, the antifungal protein (ZSU2) isolated and purified from the seed of arugula exhibited a molecular mass about 12 kDa in Tricine-SDS-PAGE electrophoresis.It had a strong inhibitory effect on 12 kinds of fungi, such as Colletotrichum higginsianum. The antifungal activity was stable on pH 1 to 13 and temperature 0 to 100℃, and had a strong metal cation tolerance.In addition, ZSU2 protein could induce chitin accumulation and cell membrane selection through sexual change.