Description: Previously we described the development of a new Staphylococcus aureus vaccine denoted MASTIVAC I. The present study describes an attempt to reduce S. aureus udder infection by MASTIVAC I vaccination in both infected and uninfected animals in a herd of 267 multiparous Israeli-Holstein cows, of which 22.1 percentage were chronically infected with S. aureus. Four groups were comprised: 1) 137 S. aureus infection-free cows and heifers that were exposed to vaccination; 2) 135 S. aureus infection-free cows and heifers that were not vaccinated and served as a control group; 3) 29 S. aureus-infected cows that were exposed to vaccination; and 4) 30 S. aureus-infected cows that were exposed to a placebo. During the 348 d of the experiment only 2/137 (1.46 percentage) immunized cows in group 1 were found to be infected with S. aureus, compared with 14/135 (10.4 percentage) cows in group 2, this difference in the new infection rate (NIR) was found significant (P < 0.05). Twenty out of 29 cows (69 percentage) in group 3 remained in the herd, compared with only 14 out of 30 cows (46.7 percentage) in group 4. Of the remaining cows, six of 20 cows (30 percentage) in the immunized group (group 3) were found to be infection-free, compared with one out of 14 cows (6.25 percentage) in the placebo group (group 4). However, this difference in the cure rate (CR) was not significant. These results strengthen findings elicited in previous experiments indicating that MASTIVACS I vaccine reduced the number of S. aureus infected animals and exerted positive effects on both the quantity and the quality of the milk production, by eliciting specific protection as well as non-specific udder health improvement. Moreover, this treatment helps to eliminate the infection from S. aureus-infected cows and reduces their somatic cell count