Effective mucosal adjuvants enhance the magnitude and quality of the vaccine response. Cyclic di-GMP (CDG) is a promising mucosal vaccine adjuvant. However, its in vivo mechanisms are unclear. Here, we showed, in mice, that CDG elicits stronger Ab and T-H responses than the mammalian 2'3'-cyclic GMP-AMP (cGAMP), and generated better protection against Streptococcus pneumoniae infection than 2'3'-cGAMP adjuvanted vaccine. We identified two in vivo mechanisms of CDG. First, intranasally administered CDG greatly enhances Ag uptake, including pinocytosis and receptor-mediated endocytosis in vivo. The enhancement depends on MPYS (STING, MITA) expression in CD11C(+) cells. Second, we found that CDG selectively activated pinocytosis-efficient-DCs, leading to T-H polarizing cytokines IL-12p70, IFN gamma, IL-5, IL-13, IL-23, and IL-6 production in vivo. Notably, CDG induces IFN lambda, but not IFN beta, in vivo. Our study revealed previously unrecognized in vivo functions of MPYS and advanced our understanding of CDG as a mucosal vaccine adjuvant.