Staphylococcus aureus is the most common etiologic agent of mastitis in bovines and a major cause of economic losses in the dairy industry. Since adhesins, that anchor bacteria to the extracellular matrix (ECM), are among the most important S. aureus virulence factors, we used four adhesion factors (fibrinogen binding protein, fibronectin binding protein A, clumping factor A and collagen adhesin) as target in a DNA vaccine. Intranasal immunization with a pDNA mixture coding the four adhesins, triggered significant levels of specific serum and mucosal Ig that inhibited S. aureus adhesion to cow mammary gland epithelial cells in vitro. Splenocytes of immunized mice challenged in vitro with S. aureus extracts showed a strong proliferative response. Finally, immunized mice were significantly protected against intramammary challenge with S. aureus. Our data confirm the feasibility of a genetic mucosal vaccine targeting S. aureus adhesins and prompt further study to test the efficacy in preventing cow mastitis.