How the central innate immune protein, STING, is activated by its ligands remains unknown. Here, using structural biology and biochemistry, we report that the metazoan second messenger 2′3′-cGAMP induces closing of the human STING homodimer and release of the STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 2′3′-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a therapeutic hypothesis for autoimmune syndrome treatment.