Microneme protein 3 (MIC3) is an important adhesion molecule expressed by Toxoplasma gondii and Neospora caninum that plays a crucial role in invasion. In our study, we found that recombinant TgMIC3 (rTgMIC3) was recognized by both T. gondii-reactive sera and hyper-immune serum against N. caninum. Polyclonal antibodies against TgMIC3 reacted with N. caninum by western blot and immunofluorescence assay (IFA). These results indicate that MIC3 is a novel cross-reactive antigen expressed in N. caninum and T. gondii. To evaluate the immune-protective effect of TgMIC3, we created the eukaryotic expression vector pcDNA3.1-TgMIC3, transfected this vector into HEK293T cells by lipofection, and evaluated TgMIC3 expression in HEK293T cells using western blot and IFA. Then, groups of BALB/c mice were immunized with recombinant TgMIC3 protein, pcDNA3.1-TgMIC3, or two-vaccine immunization. The mice were challenged with T. gondii RH or N. caninum Nc-1 tachyzoites 14 days after the final immunization. The survival time of T. gondii-infected mice was recorded, and the parasite burden in the brain of N. caninum-infected mice 30 days post-infection was measured using real-time PCR. The results demonstrated that mice immunized with TgMIC3-based vaccines elicited high antibody titers. After parasitic challenge, mice immunized with pcDNA-TgMIC3 exhibited prolonged survival when infected with T. gondii tachyzoites and a lower parasitic burden in the brains of mice challenged with N. caninum tachyzoites. These results demonstrate that TgMIC3 is a cross-protective antigen expressed in T. gondii and N. caninum and could elicit some protection against toxoplasmosis and neosporosis.