Staphylococcus aureus drug resistance to antibiotics is a serious situation that has drawn greater attention to immunotherapy and prophylaxis. Peptidoglycan (PGN) is a common and conserved component of the cell wall of Gram-positive bacteria such as S. aureus. However, PGN, as a thymus-independent antigen, cannot be considered a vaccine candidate because of its very weak immunogenicity. In this study we have attempted to enhance the immunogenicity of PGN by identifying a PGN peptide mimic sequence that would act as a thymus-dependent antigen. Several peptide sequences were obtained from a phage display peptide library using a mAb against S. aureus PGN, and a 12-mer linear single peptide (Sp-31) and a four-branch multiple antigen peptide (MAP) (MAP-P31) were synthesized. Both Sp-31 and MAP-P31 were shown to bind directly to anti-PGN mAb and a polyclonal antibody against S. aureus. These peptides could also inhibit the binding of PGN to a mAb against PGN. Furthermore, MAP-P31 was able to provoke an effective secondary antibody response in mice to PGN and to cell-wall fragments isolated from S. aureus, Escherichia coli, Staphylococcus epidermidis and Pseudomonas aeruginosa by sonication. In addition, the MAP-P31 antiserum showed a potent bactericidal or bacteriostatic activity against S. aureus in the presence and absence of complement in vitro. Importantly, immunization with MAP-P31 significantly prolonged the survival and enhanced bacterial clearance in BALB/c mice challenged with live S. aureus. In addition, the serum IgG-type antibodies against PGN persisted in mice, demonstrating that MAP-P31, as a peptide mimicking epitopes on PGN, provokes an effective secondary or memory antibody response, which can only be induced by a thymus-dependent antigen and which protects against infection with S. aureus. These results suggest that MAP-31 may be a novel vaccine candidate against S. aureus.