Staphylococcus aureus mastitis is the major cause of economic losses in dairy production worldwide. Dairy cows are very susceptible to mastitis during periparturient and early dry periods. Currently there is no effective vaccine for S. aureus mastitis. Despite adoption of hygienic milking practices and subsequent reduction in S. aureus mastitis in most well managed dairy farms, overall S. aureus mastitis continues to be the most common disease of dairy cattle. This organism usually causes chronic mastitis and treatment with antibiotics is of limited success. The objective of this study was to evaluate the immune responses and protection in dairy cows vaccinated with Staphylococcus aureus surface proteins (SASP) and Staphylococcus chromogenes surface proteins (SCSP). A total of 17 Holstein dairy cows were randomly divided into 3 groups. Cows in group 1 (n = 5) and group 2 (n = 6) were vaccinated with 1.2 mg of SASP and SCSP with an Emulsigen-D adjuvant respectively. Cows in a group 3 were injected with PBS mixed with an Emulsigen-D and served as a control. Three consecutive vaccinations were given subcutaneously in the neck area at 28 d before drying off (d −28), 14 d before drying off (d −14) and at drying off (d 0). Milk or mammary secretion and serum antibody titers were evaluated by ELISA. All cows were challenged with S. aureus strain UT2 by teat dipping in a bacterial suspension at an average cell density of approximately 10⁶ cfu/mL of growth medium. Statistical analyses were performed using GraphPad Prism version 7.03. Differences in antibody titers, number of S. aureus counts and somatic cell counts were analyzed using a one-way ANOVA, followed by a Kruskal-Wallis test. Results showed that vaccinated cows had significantly increased serum IgG, IgG1, IgG2 antibody titers compared with the control cows (P ≤ 0.05). Vaccinated cows shed reduced number of S. aureus through milk and SCSP vaccine cross-protected against S. aureus clinical mastitis.