Objective： To construct DNA vaccines expressing prostate-specific membrane antigen （PSMA） and/or granulocytemacrophage colony-stimulating factor （GM-CSF） and to determine their immunoactivity. Methods： Recombinant plasmids pIRES-PSMA-mGM-CSF , pIRES-PSMA, and pIRES-mGM-CSF were constructed with DNA vaccine vector plRES. Alter identified by endonuclease digestion, the above 3 plasmids and blank pIRES vector were used to immunize C56BL/6 mice （n= 15）. LDH release assay was used to exam the cytotoxicity of cytolytic T lyrnphocytes in each group. Results： We successfully constructed the above mentioned recombinant plasmids. Mice in pIRES-PSMA-mGM-CSF immunized group had the highest specific cytotoxicity, followed by pIRES-PSMA and pIRES-mGM-CSF immunized groups. The blank pIRES group had the lowest cytotoxicity （P〈0.05）. The cytotoxicity was the highest in all 4 groups at an effector/target ratio of 10/1. Condusion： The bicistronic DNA vaccine expressing PSMA and mGM-CSF may have a promising therapeutic value in gene therapy of prostate cancer.