In this study, we provide evidence that mammalian cells survey their cytosol for the presence of c-di-GMP. We find that c-di-GMP triggers a transcriptional response virtually indistinguishable from the response triggered by cytosolic DNA. Like the response to DNA, the response to c-di-GMP requires the TBK1 kinase and IRF3 transcription factor. However, the pathway for sensing c-di-GMP can be distinguished from that for sensing DNA in certain cell types and, moreover, appears to be distinct from all other known cytosolic sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.