Neospora caninum (N. caninum) is an apicomplexan parasite and causes abortion and congenital neosporosis in cattle worldwide. In this study, we evaluate the virulence of a N. caninum isolate on mouse strain C57BL/6. Six groups of five mice C57BL/6 were intraperitoneally inoculated with 1 × l07, 1.5 × l07, 2 × l07, 3 × l07 and 4 × l07 tachyzoites and a control inoculum of DMEM, respectively. Clinical signs and mortality rate were recorded and confirmed by histopathological findings and molecular method. The results of this study indicated that LD50 was 2.5 × l07 tachyzoites of N. caninum per C57BL/6 mouse. This can be used as a lethal challenge model in vaccine development studies.