A novel method for preparation of CpG ODN-encapsulated anionic poly(amino acid) nanoparticles (NPs) was developed for gene delivery purposes. CpG ODN was successfully encapsulated into the NPs using self-assembly of amphiphilic poly(amino acid), and the amount of encapsulated CpG ODN was dependent on the complex ratio of CpG ODN and polycation. The DNA-encapsulated anionic NPs may be useful as a gene delivery and DNA vaccine system with low toxicity.