The aim of the present study was to evaluate the seroprevalence of antibodies against Neospora caninum, obtained two years apart, among dairy cattle in a rural settlement in southern Brazil. Blood samples from 734 dairy cattle on 41 farms were collected at two different times: in 2012, 406 animals on 30 farms were used; and in 2014, 329 animals on 31 farms. Serum samples were obtained and were used to detect antibodies against N. caninum, by means of the indirect fluorescence assay (IFA). Animals with titers ? 100 were considered positive. The total serum prevalence of anti-N. caninum antibodies was 19.7% (145/736) among all the dairy cattle, comprising 23.1% (94/406) in 2012 and 15.5% (51/329) in 2014. Serum from 91 animals was tested in both trials: 11(12.1%) showed positivity in 2012 and 10 (11%) in 2014. The variables of age, sex and breed did not show any associations with seropositivity. Thus, we showed that the cattle in this settlement presented high levels of antibodies against N. caninum, and that IFA showed good efficacy for epidemiological studies.