NK4, composed of the N-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts not only as a competitive antagonist of HGF but also as an inhibitor of angiogenesis. By studying the antitumor effect of NK4, we evaluated the potential of gene therapy with NK4 as a treatment for pancreatic cancer. Expression vector pcDNA3-NK4 containing NK4 cDNA was used to transfect human pancreatic cancer cell line SUIT-2. Although the established NK4 transfectant continuously expressed NK4 protein, the expression was shown by migration assay to be insufficient to antagonize HGF in vitro. Proliferation of the NK4 transfectant did not differ significantly from that of a mock transfectant. In vivo, we used models of orthotopic implantation and liver metastasis to transplant NK4-transfected clone or mock-transfected clone into nude mice. Cell proliferation in vivo, evaluated by immunohistochemical staining of proliferating cell nuclear antigen, did not differ between NK4 and mock transfectants, and this was also the finding in the in vitro assay. However, the NK4-transfected clone showed significant inhibition of tumor progression in both the orthotopic implantation and liver metastasis models. The number of vessels within tumors was significantly decreased, and the apoptotic tumor cells were increased in number. The results of these experiments show that genetic modification of tumor cells with NK4 cDNA yields a significant antitumor effect and that this effect is mainly obtained by NK4's function as an angiogenesis inhibitor rather than as an HGF antagonist. We conclude that the potent angiogenesis inhibitor NK4 may be a promising molecule for gene therapy of pancreatic cancer.