In recent studies, heterologous prime-boost approaches, employing plasmid DNA and viral vector pathogen-delivering sequences, have been considered an effective protection strategy for intracellular parasite infections. Here, we evaluated the efficacy of such a strategy against the canine Babesia gibsoni infection. The DNA (pCAGGS-P29) and recombinant vaccinia virus (vvP29) both encoding the P29 of B. gibsoni were used in this study. The dogs were immunized 3 times with priming DNA and boosted once with recombinant virus. The dogs immunized with P29 developed a significant level of IgG2 antibody against P29. The response was strongly boosted by the inoculation of vvP29. The peripheral IFN-gamma responses of the dogs immunized with P29 were significantly higher than those of controls after the parasite inoculation. Moreover, the P29 immunized group showed a significantly low level of parasitemia. In conclusion, this study supports the efficacy of a prime-boost strategy for dogs against canine B. gibsoni infection.