The sensing of foreign DNA is crucial for host defense against several pathogens. In the cytosol the enzyme cyclic GMP-AMP synthase (cGAS) is critical for the recognition of pathogen-derived DNA and provides key signals that initiate immune responses. Beyond infection, accumulating evidence indicates that cGAS is also active during autoimmune diseases and controls immune responses that underlie cancer immunosurveillance. In this review, we highlight our current understanding of DNA sensing by cGAS and its involvement in human disease.