Neosporosis is primarily a disease of cattle and dogs, but Neospora caninum has been linked to abortion and neonatal mortality in sheep. Since the economic, clinical and epidemiological importance of the infection in sheep remains uncertain, this work investigated the seroprevalence of anti-N. caninum antibodies and associated factors in the rapidly expanding flock of Rio de Janeiro state. Blood samples from 388 sheep of 12 farms were collected and sera tested by a commercial Enzyme-Linked Immunosorbent Assay. Seroprevalence at the animallevel was of 6.2% (24/388) and, at the herd-level, 50% (6/12) of the studied farms had at least one seropositive animal. Multivariate analysis detected that occasional veterinary assistance (P < 0.05) was significantly associated to higher seroprevalence, which is also associated to age (P < 0.001) and gender (P < 0.0001). Farmers' investments should focus on making technical assistance more frequent and future studies should assess the association of veterinary assistance with anti-N. caninum antibodies in sheep flocks.