A serosurvey was carried out to estimate the herd-level seroprevalence of Neospora caninum infection in cattle in central and northeastern Poland. Ninety seven dairy cattle herds from 2 provinces of Poland (Podlaskie, 47 herds and Łodzkie, 50 herds) were randomly enrolled in the study using two-stage cluster method. A simple random selection was applied within each herd to select a sample of adult cows (≥18 month-old). A total number of 734 cows were enrolled in the study. The animals were screened with a commercial competitive ELISA (Bio-X Diagnostics, Belgium). To calculate true herd-level seroprevalence test sensitivity and specificity were adjusted from an individual- to a herd-level using FreeCalc method. The true overall herd-level seroprevalence of N. caninum infection was 56.7% (95% CI: 47.5%, 65.9%). The true herd-level seroprevalence in Podlaskie was 63.3% (95% CI: 43.0%, 83.6%) and 50.5% (95% CI: 32.8%, 68.2%) in Łodzkie province and these figures did not differ significantly between the two provinces (chi2 test p = 0.238). One hundred forty three of 734 cows (19.5%) were seropositive which gave the true overall individual-level seroprevalence of 20.1% (95% CI: 17.4%, 23.2%). Percentage of seropositive cows in each herd varied from 6% to 80%. This study is the first epidemiological investigation of herd-level seroprevalence of N. caninum infection in Polish dairy cattle population. In conclusion, the result of the study confirmed previous data that N. caninum infection is widespread in the Polish cattle population and thus should be considered as a potential cause of spontaneous abortions.