The objective of this study was to investigate the effect of a herd specific vaccine against Staphylococcus aureus (S. aureus) by different inoculation route, IMI (intramammary injection), Koso, and supramammary lymphnode area. Based on strains of S. aureus from a case of acute clinical mastitis in Daekwanryeong farm, two experimental vaccines were made, inactivated vaccine and sonicated one. To determine the antigen concentration of the vaccine, 220 ICR-mice were immunized by the intraperitoneal route with inactivated or sonicated experimental vaccines according to different schedules with 10 ICR mice as a control group. The sonicated vaccine was selected for further experiments in vaccination and the highest antibody titer in ELISA was observed at 3㎎/㎖ of the vaccine. The vaccine was administrated to 22 healthy cows at drying up. The quarter milk samples and bloods were collected before vaccination, right after parturition and 3 months postpartum. There were no significant differences among vaccination routes based on the antibody titers in serum and intramammary bacterial infections during drying up period. The antibody titer in serum of vaccinated cows was higher at parturition than 3 months postpartum but it had no statistical meaning, though decreased clinical signs and morbidity were observed. Results of this study suggest that the concept of the vaccine against S. aureus is to decreasing clinical mastitis rather than preventing the disease.