The micronemal protein MIC3 of Toxoplasma gondii plays a predominant role in the early phase of the invasion process. In this research, the expression and location of protein MIC3 and fusion protein EGFP-MIC3 were observed in host cells and in T. gondii respectively. Protective experiments of DNA vaccine pcDNA3-MIC3 (pMIC3i) in animals showed that the vaccine could significantly prolong the survival time of the mice challenged by virulent RH strains of T. gondii. All mice vaccinated with plasmid pcDNA3-MIC3 have been elicited specific humoral immunity and cellular immunity. The cellular immune response was associated with the increase of the CD4(+) and CD8(+) T lymphocytes and the decrease of the CD4(+)/CD8(+) T lymphocyte ratio evidently. It indicated that the mic3 DNA vaccine could stimulate the host resisting Toxoplasma mainly by the way of CD8(+)CTL cells. In conclusion, a potent DNA vaccine pcDNA3-MIC3 could elicit a strong specific immune response and induce effective protection against T. gondii challenge in Kunming mice, suggesting that mic3 is a potential vaccine candidate against toxoplasmosis.