We investigated the ability of NK4, an antagonist of human hepatocyte growth factor (HGF), to inhibit the influence of HGF on proliferation, migration, invasion and apoptosis of human prostate cancer cells. Expression vector pBudCE4.1-EGFP-NK4 containing NK4 cDNA was used to transfect human prostate cancer DU145 cells, and the effects of the autocrine NK4 on tumor cell proliferation, migration, invasion and apoptosis were assessed in vitro. In vivo, we subcutaneously implanted DU145 cells, mock-transfected clone (DU145/empty vector) cells and NK4-transfected clone (DU145/NK4) cells into nude mice, and then evaluated tumor growth, cell proliferation and cell apoptosis in vivo. We found that DU145/NK4 cells expressed NK4 protein. In the in vitro study, autocrine NK4 attenuated the HGF-induced tumor cell proliferation, migration and invasion, and stimulated apoptosis. Furthermore, autocrine NK4 effectively inhibited the HGF-induced phosphorylation of c-Met, extracellular signal-regulated kinase-1 (ERK1). and protein kinase B 1/2 (Akt1/2). Histological examination revealed that autocrine NK4 inhibited proliferation and accelerated apoptosis of prostate cancer cells. These results show that genetic modification of DU145 cells with NK4 cDNA yields a significant effect on their proliferation, migration, invasion and apoptosis. Molecular targeting of HGF/c-Met by NK4 could be applied as a novel therapeutic approach to prostate cancer.