Cyclic diguanylate (c-di-GMP) is a novel immunomodulator and immune enhancer that triggers a protective host innate immune response. The protective effect of c-di-GMP as a vaccine adjuvant against Staphylococcus aureus infection was investigated by subcutaneous (s.c.) vaccination with two different S. aureus antigens, clumping factor A (ClfA) and a nontoxic mutant staphylococcal enterotoxin C (mSEC), then intravenous (i.v.) challenge with viable methicillin-resistant S. aureus (MRSA) in a systemic infection model. Mice immunized with c-di-GMP plus mSEC or c-di-GMP plus ClfA vaccines then challenged with MRSA produced strong antigen-specific antibody responses demonstrating immunogenicity of the vaccines. Bacterial counts in the spleen and liver of c-di-GMP plus mSEC and c-di-GMP plus ClfA-immunized mice were significantly lower than those of control mice (P<0.001). Mice immunized with c-di-GMP plus mSEC or c-di-GMP plus ClfA showed significantly higher survival rates at day 7 (87.5%) than those of the non-immunized control mice (33.3%) (P<0.05). Furthermore, immunization of mice with c-di-GMP plus mSEC or c-di-GMP plus ClfA induced not only very high titers of immunoglobulin G1 (IgG1), but c-di-GMP plus mSEC also induced significantly higher levels of IgG2a, IgG2b and IgG3 compared to alum adjuvant (P<0.01 and P<0.001, respectively) and c-di-GMP plus ClfA induced significantly higher levels of IgG2a, IgG2b and IgG3 compared to alum adjuvant (P<0.001). Our results show that c-di-GMP should be developed as an adjuvant and immunotherapeutic to provide protection against systemic infection caused by S. aureus (MRSA).