Diphtheria toxin is an exotoxin secreted by Corynebacterium diphtheriae that has been lysogenized by β bacteriophage that carries the DT gene. It blocks protein synthesis and kills the target eukaryotic cell. The R82A ， K84A ， H86A mutant of vascular endothelial growth factor (VEGF) specially binds its receptor 1 (VEGFR-1) that is expressed highly on surface of tumor blood vessel. DT genomic DNA was extracted first and then the gene that coding the T domain and C domain of DT (DT391) were amplified. The R82A ， K84A ， H86A mutant were introduced to VEGF by site-directed mutagenesis. Then a VEGFR-1 targeting fusion protein, DT391-mVEGF, was constructed by substituting the receptor binding domain of DT with the VEGF mutant which shows high affinity to a receptor expressed on tumor vascular. With DT391, a protein without the mVEGF domain of DT391-mVEGF, as a negative control in cytotoxicity assay, the hybrid protein DT391-mVEGF showed an inhibition to the growth of VEGFR-1 positive tumor cell.