Host cell invasion by Toxoplasma gondii is tightly coupled to the apical release of micronemal proteins (MIC). In this work, we evaluated the protective effect encountered in C57BL/6 mice immunized with MIC1 and MIC4 purified from soluble tachyzoite antigens by affinity to immobilized lactose. The immunized mice presented high serum levels of IgG1 and IgG2b specific antibodies. MIC1/4-stimulated spleen cells from immunized mice produced IL-2, IL-12, IFN-gamma, IL-10, but not IL-4, suggesting the induction of a polarized Th1 type immune response. When orally challenged with 40 cysts of the ME49 strain, the immunized mice had 68% fewer brain cysts than the control mice. Immunization was associated with 80% survival of the mice challenged with 80 cysts, contrasting with 100% mortality of the non-immunized mice in the acute phase. In this phase, there was much lower parasitism in the lungs and small intestine of the immunized mice, and they did not exhibit the early-stage signs of intestinal necrosis, which was clearly detected in the control mice. Our data demonstrate that MIC1 and MIC4 triggered a protective response against toxoplasmosis, and that these antigens are targets for the further development of a vaccine.