The innate immune system utilizes pattern-recognition receptors (PRRs) to detect the invasion of pathogens and initiate host antimicrobial responses such as the production of type I interferons and proinflammatory cytokines. Nucleic acids, which are essential genetic information carriers for all living organisms including viral, bacterial, and eukaryotic pathogens, are major structures detected by the innate immune system. However, inappropriate detection of self nucleic acids can result in autoimmune diseases. PRRs that recognize nucleic acids in cells include several endosomal members of the Toll-like receptor family and several cytosolic sensors for DNA and RNA. Here, we review the recent advances in understanding the mechanism of nucleic acid sensing and signaling in the cytosol of mammalian cells as well as the emerging role of cytosolic nucleic acids in autoimmunity.