Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-α, IFN-γ, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-γ was not observed. The mean cytokine released after 24 and 72h of infection were 3.8±0.6 and 3.7±0.6pg TNF-α/mg protein and 2.7±0.69 and 4.1±0.64pg IL-10/mg protein, respectively, and more than 8.0pg IL-6/mg protein for both time points. NO levels increased 24h post-infection (2.3±0.8pg/mg protein) until 72h (4.2±1.1pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-γ; high levels of TNF-α and NO may represent an effective response by infected glial cells against N. caninum.