The bacterial second messenger (3'-5')-cyclic-di-guanosine-monophosphate (CDG) is a promising mucosal adjuvant candidate that activates balanced Th1/Th2/Th17 responses. We showed previously that CDG activates stimulator of IFN genes (STING)-dependent IFN-I production in vitro. However, it is unknown whether STING or IFN-I is required for the CDG adjuvant activity in vivo. In this study, we show that STING(-/-) mice (Tmem173(<tm1Camb>)) do not produce Ag-specific Abs or Th1/Th2/Th17 cytokines during CDG/Ag immunization. Intranasal administration of CDG did not induce TNF-α, IL-1β, IL-6, IL-12, or MCP-1 production in STING(-/-) mice. Surprisingly, we found that the cytokine and Ab responses were unaltered in CDG/Ag-immunized IFNAR(-/-) mice. Instead, we found that CDG activates STING-dependent, IFN-I-independent TNF-α production in vivo and in vitro. Furthermore, using a TNFR1(-/-) mouse, we demonstrate that TNF-α signaling is critical for CDG-induced Ag-specific Ab and Th1/Th2 cytokine production. This is distinct from STING-mediated DNA adjuvant activity, which requires IFN-I, but not TNF-α, production. Finally, we found that CDG activates STING-dependent, but IRF3 stimulation-independent, NF-κB signaling. Our results established an essential role for STING-mediated TNF-α production in the mucosal adjuvant activity of CDG in vivo and revealed a novel IFN-I stimulation-independent STING-NF-κB-TNF-α pathway.