The inappropriate presence of DNA in the cytosol is a danger signal that alerts the host of potential microbial invasion and triggers innate immune responses including the production of type-I interferons. Under certain pathological conditions, self DNA, which normally reside in the nucleus or mitochondria, could also trigger autoimmune responses from the cytosol, resulting in human diseases such as lupus. Cytosolic DNA induces interferons through a signaling pathway that involves the adaptor protein STING, the kinases IKK and TBK1, and the transcription factors NF-kappaB and IRF3. Through a biochemical approach, we have identified the cytosolic DNA sensor that activates STING and triggers type-I interferon production. This sensor turns out to be a novel enzyme which we name cyclic GMP-AMP synthase (cGAS). cGAS is activated by its binding to DNA and upon activation it catalyzes the synthesis of a unique cyclic GMP-AMP (cGAMP) isomer containing both 2’-5’ and 3’-5’ phosphodiester linkages. This cGAMP isomer, termed 2’3’-cGAMP, functions as a second messenger that binds to and activates STING, leading to the induction of interferons and other cytokines. Genetic experiments show that cGAS is essential for innate immune responses triggered by DNA viruses and retroviruses, including HIV. This work uncovers a cyclic dinucleotide signaling pathway that was previously not known to exist in metazoa, reveals a new signaling mechanism in innate immunity, provides cGAS as a new therapeutic target for the treatment of autoimmune diseases, and offers cGAMP as a candidate molecule for the development of new vaccines and immune therapeutics.