The innate immune system is the first line of defense against invading pathogens. One important feature of innate immune recognition is self versus nonself discrimination. The selectivity for microbial ligands is achieved through substrate motif specificity, spatial compartmentalization, and functions of negative regulators. Loss-of-function mutations in negative regulators or gain-of-function mutations in drivers of innate immune signaling have been associated with autoimmune diseases such as lupus, rheumatoid arthritis, inflammatory vasculopathy, and a variety of interferonopathies. This review will focus on TREX1 and STING, which are opposing regulators of the cytosolic DNA-sensing pathway. Tremendous effort over the past decade among academic and clinical research groups has elucidated molecular mechanisms underlying immune diseases associated with TREX1 and STING dysfunction. We have also witnessed rapid therapeutic translation of the molecular findings. Several targeted treatment options or druggable candidates are now available for these once incurable diseases. With great enthusiasm from both academia and industry partners, we look forward to seeing the remaining scientific questions answered and, more importantly, the affected patients benefited from these discoveries.