Staphylococcus aureus infections are becoming a major global health issue due to the rapid emergence of multidrug-resistant strains. Therefore, there is an urgent need to develop an effective vaccine to prevent and control these infections. In order to develop a universal immunization strategy, we constructed a mutant derivative of S. aureus 132 which lacks the genes involved in D-alanine biosynthesis, a structural component of cell wall peptidoglycan. This unmarked deletion mutant requires the exogenous addition of D-alanine for in vitro growth. The aim of this study was to examine the ability of this D-alanine auxotroph to induce protective immunity against staphylococcal infection. Our findings demonstrate that this deletion mutant is highly attenuated, elicits a protective immune response in mice and generates cross-reactive antibodies. Moreover, the D-alanine auxotroph was completely eliminated from the blood of mice after its intravenous or intraperitoneal injection. We determined that the protective effect was dependent on antibody production since the adoptive transfer of immune serum into naïve mice resulted in effective protection against S. aureus bacteremia. In addition, splenocytes from mice immunized with the D-alanine auxotroph vaccine showed specific production of IL-17A after ex vivo stimulation. We conclude that this D-alanine auxotroph protects mice efficiently against virulent staphylococcal strains through the combined action of antibodies and IL-17A, and therefore constitutes a promising vaccine candidate against staphylococcal disease, for which no licensed vaccine is available yet.