Prostate cancer is one of the most prevalent tumors. The switch of androgen signal dependence makes therapy more complex. Although reports on introduction of a single suicide gene exist, double suicide gene therapy has not been reported yet. In the current study, two suicide genes were constructed in the pIRES plasmid driven by PSMA promoter. 5-FC and GCV combination in vitro led to a higher growth inhibition on prostate cancer compared to a single pro-drug. Retarded xenograft tumor growth was observed in castrated nude mice after double suicide gene activation. Furthermore, decreased metastasis was observed with double suicide gene treatment. These findings suggest that specific double suicide gene strategy could be a potential option for the therapy of prostate cancer.