NK4, composed of the NH2-terminal hairpin and subsequent four-kringle domains of hepatocyte growth factor (HGF), acts as a potent angiogenesis inhibitor. This study is an investigation to evaluate the feasibility of controlled release of NK4 plasmid DNA in suppressing tumor growth. Controlled release by a biodegradable hydrogel enabled the NK4 plasmid DNA to enhance the tumor suppression effects. Biodegradable microspheres of cationized gelatin were prepared for the controlled release of a NK4 plasmid DNA. The cationized gelatin microspheres incorporating NK4 plasmid DNA were subcutaneously injected to tumor-bearing mice to evaluate the suppressive effects on tumor angiogenesis and growth. The cationized gelatin microspheres incorporating NK4 plasmid DNA could release over 28 days. When the cationized gelatin microspheres incorporating NK4 plasmid DNA were injected into the subcutaneous tissue of mice intraperitoneally inoculated with pancreatic cancer cells, their survival time period was prolonged. Tumor growth was suppressed to a significantly greater extent than free NK4 plasmid DNA. The controlled release of NK4 plasmid DNA suppressed angiogenesis and increased cell apoptosis in the tumor tissue, while it enhanced and prolonged the serum level of NK4 protein. We conclude that the controlled release technology was promising to enhance the tumor suppression effects of NK4 plasmid DNA.