Neospora caninum is an intracellular protozoan pathogen that causes abortion in cattle. This parasite elicits a typical type 1 immune response in host animals, and it is widely believed that the strong type 1 immune response during pregnancy may result in fetal death. Pro-inflammatory and/or inflammatory cytokines produced during either primary or secondary pathogen exposure are supposed to be the mediators of abortion. The present study defined cytokine production by murine naïve dendritic cells and spleen cells in response to whole Neospora tachyzoites (live, heat-killed, freeze-killed) or whole-cell tachyzoite lysate in the form of total (NcAg), soluble (sNcAg), or insoluble antigen (isNcAg). All tachyzoite and antigen preparations at high doses stimulated high levels of interleukin (IL) -12, interferon (IFN) -γ, and tumor necrosis factor (TNF) -α, except for heat-killed tachyzoites and sNcAg, which induced moderate level of IL-12 and very low levels of IFN-γ and TNF-α. In general, whole N. caninum tachyzoites were more effective in inducing IL-12, IFN-γ, and TNF-α than the lysate antigen preparations. It appears that the heat-killed N. caninum tachyzoites were less potent in eliciting IFN-γ or IL-10, but more effective in inducing IL-4. Thus, heat-inactivated tachyzoites or sNcAg alone may not be powerful enough to elicit strong type 1 immune responses against the disease. The present study comprehensively studied the production of critical cytokine by the murine dendritic cells and spleen cells in response to N. caninum; these results may facilitate a better understanding of antigen priming and aid in the design of vaccines/adjuvants against neosporosis.