3′, 5′-Cyclic diguanylic acid (c-di-GMP) is a bacterial intracellular signaling molecule that plays a crucial role in the regulation of bacterial motility, adhesion, cell-to-cell communication, exopolysaccharide synthesis, biofilm formation and virulence. The recent finding that c-di-GMP can act as a danger signal on eukaryotic cells has prompted the study of the immunostimulatory and immunomodulatory properties of c-di-GMP in an effort to determine whether c-di-GMP might be further developed as a potential vaccine adjuvant. In this review, we discussed the recent in vitro and in vivo studies of the immunostimulatory properties of c-di-GMP and the progress that has been made in the preclinical development of c-di-GMP as a potential vaccine adjuvant for systemic and mucosal vaccination.