The protection conferred by antiviral DNA vaccines in fish is known to rely greatly on innate immune responses. Since oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides (CpG motifs) have been shown to induce potential innate immune responses, we have introduced several copies (either two or four) of a fragment containing multiple CpG sequences of known immunostimulatory effects into a DNA vaccine against viral hemorrhagic septicemia virus (VHSV). We have determined the effects of this introduction on the vaccine immunogenicity, measured as immune gene induction, serum neutralizing activity and antigen-dependent proliferation. When comparing the effects of the vaccine containing 2 copies of this CpG fragment (pVHSV-2CpG) or that containing 4 copies of the fragment (pVHSV-4CpG) with the original VHSV DNA vaccine (pVHSV), we found that the levels of expression of type I interferon (IFN) were significantly up-regulated in muscle and spleen when the CpG fragments were introduced. An up-regulation in the levels of MHC-I expression in spleen were also observed in response to the modified vaccines, whereas, the levels of transcription of interleukin 1β (IL-1β) were strongly reduced in comparison to the original vaccine. Important but very variable differences were also observed concerning the vaccine induction of IFN-γ. Moreover, the serum neutralizing capacity was strongly increased as fish were vaccinated with plasmids containing more CpG fragments. Taken together, all these results demonstrate a strongly increased immunogenicity of the VHSV DNA vaccine, through the introduction of this multicopy CpG fragment.