HER2/neu is an attractive target for tumor therapy since its overexpression in a number of tumors. In order to enhance the cytotoxicity of anti-HER2/neu antibody, and the specificity of TNF-α for tumor cell, a fusion gene of antiHER2-hTNF-α was constructed. The recombinant fusion protein was produced in E.coli, and their refolding has done through affinity chromatography on a His-column. The ELISA assay showed that antiHER2-hTNF-α specifically bound to HER2/neu expressing SKOV-3 and MCF-7 cells, but it did not recognize the HER2/neu negative cells A375. In vitro study, it was found that antiHER2-hTNF-α inhibited the proliferation of SKOV-3 and MCF-7 cells, whereas it did not effect on the A375. These studies suggest that the recombinant antiHER2-hTNF-α immunotoxin has a potential application in HER2/neu expressing tumor therapy.