LHRH receptor, is over-expressed in a variety of human tumors and, is a potential binding site for targeted metastatic prostate cancer therapy. The objectives of our study were to synthesize a bioconjugate of the LHRH analog [DLys6]-LHRH and the anti-tumor agent methotrexate and test the hypothesis that [DLys6]-LHRH-MTX targets and inhibits prostate cancer cell growth in vitro and in vivo. The results of in vitro studies, showed that both [DLys6]-LHRH-MTX and MTX displayed superior cytotoxicity against prostate cancer cells in a concentration-dependent manners, with IC50 concentrations for PC-3 cells of, 1.02 ± 0.18 μmol/L and 6.34 ± 1.01 μmol/L; for DU-145 cells, 1.53 ± 0.27 μmol/L and 8.03 ± 1.29 μmol/L; and for LNCaP cells, 1.93 ± 0.19 μmol/L and 9.68 ± 1.24 μmol/L, respectively. The IC50 values of [DLys6]-LHRH-MTX and MTX were 110.77 ± 15.31 μmol/L and 42.33 ± 7.25 μmol/L, respectively. Finally, [DLys6]-LHRH-MTX significantly improved the anti-tumor activity of MTX in nude mice bearing PC-3 tumor xenografts. The inhibition ratios of tumor volume and tumor weight in the [DLys6]-LHRH-MTX treated group were significantly higher than those in the MTX-treated group. Tumor volume doubling time was also significantly extended from 6.13 days in control animals to 9.67 days in mice treated with [DLys6]-LHRH-MTX. In conclusion, [DLys6]-LHRH -MTX may be useful in treating prostate cancer.