Toxoplasma gondii is distributed worldwide and infects most species of warm-blooded animals, including humans. The heavy incidence and severe or lethal damage caused by T. gondii infection clearly indicates the need for the development of a vaccine. To evaluate the protective efficacy of a multiantigenic DNA vaccine expressing GRA7 and ROP1 of T. gondii with or without a plasmid encoding murine interleukin-12 (pIL12), we constructed DNA vaccines using the eukaryotic plasmids pGRA7, pROP1, and pGRA7-ROP1. Mice immunized with pGRA7, pROP1, or pGRA7-ROP1 showed significantly increased serum IgG2a titers; production of gamma interferon (IFN-γ), IL-10, and tumor necrosis factor alpha (TNF-α); in vitro T cell proliferation; and survival, as well as decreased cyst burdens in the brain, compared to mice immunized with either the empty plasmid, pIL12, or vector with pIL12 (vector+pIL12). Moreover, mice immunized with the multiantigenic DNA vaccine pGRA7-ROP1 had higher IgG2a titers, production of IFN-γ and TNF-α, survival time, and cyst reduction rate compared to those of mice vaccinated with either pGRA7 or pROP1 alone. Furthermore, mice immunized with either a pGRA7-ROP1+pIL12 or a single-gene vaccine combined with pIL12 showed greater Th1 immune response and protective efficacy than the single-gene-vaccinated groups. Our data suggest that the multiantigenic DNA antigen pGRA7-ROP1 was more effective in stimulating host protective immune responses than separately injected single antigens, and that IL-12 serves as a good DNA adjuvant.