Neospora caninum is an important cause of abortion in cattle worldwide, but the isolation of a viable parasite from an abortus is difficult, and viable N. caninum has not been isolated from any host in China. In the present study, peripheral blood samples were collected from a jugular vein of an adult dairy cow that had aborted; the cow was seropositive to N. caninum antibodies by ELISA. White blood cells were separated and seeded onto Vero cell monolayer cultures for parasite isolation. Tachyzoites were first observed in cell culture on day 84 after initial inoculation. The parasite was confirmed to be N. caninum by gene sequencing and immunofluorescence, and by bioassays in BALB/c mice. The new N. caninum isolate (NC-Bj) has a unique pattern on microsatellite Cont-14. To our knowledge, this is the first successful isolation of N. caninum in China from any host.