Aims To evaluate the effect of a DNA priming and protein boosting immunization scheme in ducks. Methods and results Pekin ducks were immunized with pTCY/VP2 DNA vaccine; on day 14 (D14) after primary immunization, the ducks were boosted with either the same vaccine (DNA + DNA) or the rVP2 vaccine (DNA + rVP2). CpG oligodeoxynucleotides containing three copies of GACGTT motifs were used as the adjuvant in the vaccines. Compared with unimmunized controls, both immunization schemes significantly increased the titre of antigen-specific antibodies, lymphocyte proliferation index, percentage of CD4(+) and CD8(+) cells in peripheral blood mononuclear cells (PBMCs) and mRNA expression of interferon (IFN)-alpha, IFN-gamma, interleukin (IL)-6 and IL-12 in antigen-stimulated PBMCs. Furthermore, compared with the DNA + DNA homologous scheme, the DNA + rVP2 heterologous scheme significantly increased lymphocyte proliferation, percentage of CD4(+) and CD8(+) cells in PBMCs and upregulation of mRNA expression of cytokines 2 weeks after the boost (D28). Conclusions The DNA + rVP2 immunization scheme enhanced immune responses, mainly Th1 type, against parvovirus in ducks. Significance and Impact of the Study The DNA priming and protein boosting heterologous immunization strategy can be applied to develop vaccines against viral infections in ducks. It can potentially be used in breeding ducks because of long-term immunity may confer protection for ducklings.