Toxoplasma gondiiinfection occurs commonly in humans and other warm-blooded animals. Its serious impact on public health and livestock sectors makes the development of an effective vaccine particularly important. In the current study, we constructed a multiantigenic DNA vaccine expressing ROP16 and GRA7 of T. gondiiand evaluated the protective efficacy of these two fragments with or without a plasmid encoding murine costimulatory molecule B7-2. These recombinant eukaryotic expression plasmids were termed pROP16, pGRA7, pROP16-GRA7 and pB7-2, respectively. After intramuscular immunization in Kunming mice, we assessed the immune response using cytokine and antibody determinations, T lymphocyte subsets analysis, and the survival times of mice post acute T. gondiichallenge. The results showed that mice immunized with the multiantigenic DNA vaccine pROP16–GRA7 gained higher levels of IgG titers and IgG2a subclass titers, production of IFN-γ, percentage of CD8+T cells and median survival times against the acute infection of T. gondiicompared with those of mice administered with pROP16 or pGRA7 and those in control groups. Moreover, the adjuvant pB7-2 formulated with DNA vaccine boosted these humoral and cellular (Th1, CD8+T cell) immune responses. Therefore, it might be a promising genetic adjuvant to DNA vaccine against T. gondiifor further investigation.