Recent approvals of gene therapies by the FDA and the EMA for treatment of inherited disorders have further opened the door for assessment of nucleic acid pharmaceuticals for clinical usage. Arising from presence of damaged or inappropriate DNA, cancer is a condition particularly suitable for genetic intervention. The RALA peptide has been shown to be a potent non-viral delivery platform for nucleic acids. This study reports that complexation of RALA with a plasmid encoding inducible nitric oxide synthase (iNOS) DNA produces functional cationic nanoparticles with gene expression in PC-3 prostate cancer cells. Furthermore, repeated administrations of RALA/DNA nanoparticles to immunocompetent mice did not produce an immunological response, be that neutralization of the vector or release of inflammatory mediators. RALA/CMV-iNOS reduced the clonogenicity of PC-3 cells in vitro, and in an in vivo model of prostate cancer metastasis, systemically-delivered RALA/CMV-iNOS significantly improved the survival of mice. These results further validate RALA as a genetic cargo delivery vehicle and iNOS as a potent therapy for the treatment of cancer.