Live vaccination is the most protective method against bovine neosporosis, which is the major cause of bovine abortion globally. Here, the Neospora caninum parenteral strain Nc1 and NcGRA7-deficient N. caninum (NcGRA7KO), which is less virulent in mice, were evaluated as potential live vaccines. BALB/c mice were subcutaneously inoculated with high (1 �� 10) or low (1 �� 10) doses of tachyzoites. At high doses, Nc1-inoculated female mice presented decreased body weight gain and increased clinical signs and died before challenge infection with green fluorescent protein (GFP)-expressing Nc1 (Nc1-GFP), whereas NcGRA7KO-inoculated animals exhibited increased survival before and after challenge infection. Although inoculation of female mice with Nc1 or NcGRA7KO resulted in a lower brain parasite number of challenged Nc1-GFP than in noninoculated animals, the total brain parasite burden in NcGRA7KO-infected mice decreased compared with that in Nc1-infetced animals. At low dose of NcGRA7KO, increased survival rates of mice and lower total brain parasite number were observed compared with high dose of NcGRA7KO. In male mice, a significant lower brain parasite burden of Nc1-GFP was observed in both high and low doses of NcGRA7-inoculated mice, and the total parasite number in the brains of low dose of NcGRA7KO-inoculated animals was lower than that in the brains of high dose of NcGRA7KO-inoculated or noninoculated animals. In conclusion, these results suggest that NcGRA7KO parasites have potential for use as a live vaccine against N. caninum infection.