Balb/c mice were inoculated intraperitoneally (i.p.) with either 5x10^6 live virulent (group 1) or 5x10^6 live attenuated (group 2) tachyzoites, or Vero cells (group 3). Animals were sacrificed at 0, 14, 28 and 42 days post-inoculation (p.i.), with the remaining mice receiving a lethal challenge on day 48 p.i. Serum, spleen and brain samples were collected post mortem to examine humoral and cell mediated immune (CMI) responses as well as pathological lesions and to quantify parasite loads. On day 14 p.i. group 2 (attenuated) demonstrated statistically significant (P<0.001) lower levels of mean morbidity and weight loss, while also showing significantly (P=0.01) higher levels of splenocyte proliferation and IFN-γ production (P=0.003), compared to group 1 (virulent). Histology of brain samples showed milder lesions and a lower incidence of positive immunohistochemistry, demonstrating tachyzoites and tissue cysts, and statistically significant (P=0.03) lower mean burdens of parasite DNA in group 2 (attenuated) compared to group 1 (virulent). No mice from group 1 (virulent) survived beyond day 24 p.i. so it was not possible to perform group analyses after day 14 p.i. However, samples from group 2 (attenuated) were collected at days 28 and 42 p.i. and analysed for humoral and CMI responses. Control group 3 mice only demonstrated parasite-specific immune responses and positive Neospora PCR results following the challenge inoculation.