Abstract A recently described new Staphylococcus aureus vaccine “MASTIVAC I” (Patent no. PTC/IL98/00627) against S. aureus udder infection elicited protection against experimentally induced infection in cows. In the present paper we describe a large-scale vaccination field trial. A total of 452 Israeli Holstein heifers were included in the study over two consecutive years. Approximately half of the heifers (228) were vaccinated while the others (224) served as a control group. Antibody response was detected in all vaccinated animals 4–5 weeks post-primary immunization and it was sustained throughout the experimental period (300–330 days). S. aureus infection could be detected in only 3 out of 228 animals (1.3%) in the vaccinated group and in 6 out of 224 (2.7%) in the control group. These numbers were too low to be statistically evaluated. However, when somatic cell counts (SCC) and milk yields were considered, a significant difference was found between the two groups, namely, the vaccinated cows in first and second lactation had 42 and 54%, respectively, lower SCCs and milk yields 0.5 kg per day higher than the non-vaccinated control cows. These results suggest that the new vaccine elicits a non-specific health improvement of the udder in addition to specific protection against S. aureus.