This paper investigates the role of specific immune factors on the course of infection in genetic knockout (gko) mice infected with 3 different strains of Neospora caninum. Survival time and parasite persistence were examined in interferon-γ (IFN-γ), tumor necrosis factor receptor-2 (TNFR2), interleukin 10 (IL-10), beta 2 microglobulin (β2M), and inducible nitric oxide synthase (iNOS2) gko or wild-type (wt) mice following infection with either pathogenic (NC-1 or NC-2) or attenuated (NCts-8) N. caninum strains. Infection with NC-1 was 100% lethal in IFN-γ gko mice, as evidenced by mean survival times of 10–13 days, depending on the challenge dose used. TNFR2 and β2M gko mice infected with NC-1 or NC-2 strain demonstrated partial susceptibility to disease, as evidenced by histopathology and survival curves. TNFR2 or β2M gko mice were not susceptible to infection with NCts-8, on the basis of absence of pathology and lack of mortality. Lack of mortality and minimal histopathology scores demonstrated that NC-1, NC-2, and NCts-8 infections were avirulent in IL-10 and iNOS2 gko mice. Adoptive transfer of immune cells from NCts-8 vaccinated normal syngeneic mice into IFN-γ gko mice significantly (P < 0.05) prolonged mean survival times at all 3 challenge doses of NC-1 but failed to protect against mortality. Interestingly, there was a notable change in the tissue tropism of tachyzoites from the lung and brain in immunocompetent wt, TNFR2 gko, IL-10 gko, β2M gko, and iNOS2 gko mice to the liver and spleen in IFN-γ gko mice when challenged with N. caninum. On the basis of these results in gko mice, IFN-γ is a critical cytokine in the host response against acute neosporosis.