To investigate the strategy of using a multivalent polyprotein DNA vaccine against Staphylococcus aureus, a series of plasmids was used to immunize mice followed by infectious challenge. The plasmid vaccines expressed Clumping factor A (Clfa), fibronectin binding protein A (FnBPA) and the enzyme Sortase (Srt) as single proteins or combined as a polyprotein. All animals produced a mixed Th1 and Th2 response including functional antigen-specific, mostly IgG2a antibodies, sustained production of IFN-γ and a predominantly CD8+ T-cell response. Upon challenge with a virulent S. aureus isolate (Sa042), after 21 days, 55% of the multi-gene vaccinated mice survived infection compared to only 15% of the control groups. Vaccinated mice showed no signs of arthritis when challenged with the less virulent “Newman” strain that caused reactive arthritis in the controls. The results suggest that a multi-gene polyprotein-expressing nucleic acid vaccine alone produces a combined Th1 and Th2 response that can contribute to protection against the complex pathogenesis of S. aureus.