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Aims: Staphylococcus aureus is an opportunistic pathogen of humans. No commercial vaccine is available to combat S. aureus infections. In this study, we have investigated the protective immune response generated by S. aureus non‐covalently associated cell wall surface protein N‐acetylmuramoyl‐L‐alanine amidase (AM) in combination with Alum (Al) and heat‐killed S. aureus (hkSA) using murine models. Methods and Results: BALB/c mice were immunized with increasing concentrations of AM antigen or hkSA to determine their optimum concentration for vaccination. Fifty micrograms of AM and hkSA each were found to generate maximum anti‐AM IgG antibody production. BALB/c mice were immunized next with 50 µg of AM, 50 µg of hKSA and 1 mg Al vaccine formulation. Vaccine efficacy was validated by challenging immunized BALB/c mice with S. aureus Newman and three clinical methicillin‐resistant S. aureus strains. AM‐hkSA‐Al–immunized mice generated high anti‐AM IgG antibody response with IgG1 and IgG2b as the predominant immunoglobulin subtypes. Increased survival (60%–90%) with decreased clinical disease symptoms was observed in the vaccinated BALB/c mice group. A significantly lower bacterial load and decreased kidney abscess formation was observed following the challenge with S. aureus in the vaccinated BALB/c mice group. Furthermore, the efficacy of AM‐hkSA‐Al vaccine was also validated using C57 BL/6 and Swiss albino mice. Conclusions: Using murine infection models, we have demonstrated that AM‐hkSA‐Al vaccine would be effective in preventing S. aureus infections. Significance and Impact of Study: AM‐hkSA‐Al vaccine elicited strong immune response and may be considered for future vaccine design against S. aureus infections. [ABSTRACT FROM AUTHOR]