Here we demonstrated that bis-(3′, 5′)-cyclic dimeric inosine monophosphate (c-di-IMP) exhibits potent adjuvant properties. BALB/c or C57BL/6 mice were immunized with the model antigens beta-galactosidase (β-Gal) or Ovalbumin (OVA) alone or co-administered with c-di-IMP by the intranasal route. Animals receiving c-di-IMP showed significantly higher anti-β-Gal or OVA immunoglobulin G titres (IgG) in sera than those vaccinated with β-Gal or OVA alone. Furthermore, strong local immune responses were also detectable in different mucosal territories, as shown by the high levels of β-Gal-specific secretory IgA (sIgA). The analysis of the antigen-specific IgG isotypes in sera, together with the profiles of the cytokines and chemokines secreted by lymphocytes from vaccinated animals showed that the use of c-di-IMP resulted in stimulation of a mixed T_H1/T_H2/T_H17 response. Mucosal immunization of C57BL/6 mice with OVA using c-di-IMP as adjuvant also led to the stimulation of both humoral and cellular (i.e., 60% of antigen-specific lysis by in vivo CTL) responses. Our results demonstrated that the novel compound c-di-IMP exhibits strong adjuvant properties when co-administered with an antigen by the mucosal route, thereby representing a promising candidate adjuvant for the development of mucosal vaccination strategies.