Although it has been appreciated for some years that cytosolic DNA is immune stimulatory, it is only in the past five years that the molecular basis of DNA sensing by the innate immune system has begun to be revealed. In particular it has been described how DNA induces type I interferon, central in antiviral responses and a mediator of autoimmunity. To date more than ten cytosolic receptors of DNA have been proposed, but STING is a key adaptor protein for most DNA-sensing pathways, and we are now beginning to understand the signaling mechanisms for STING. In this review we describe the recent progress in understanding signaling mechanisms activated by DNA and the relevance of DNA sensing to pathogen responses and autoimmunity. We highlight new insights gained into how and why the immune system responds to both pathogen and self DNA and define important questions that now need to be addressed in the field of innate immune activation by DNA.