The recognition of cytosolic nucleic acid triggers the DNA/RNA sensor–IRF3 axis-mediated production of type I interferons (IFNs), which are essential for antiviral immune responses. However, the inappropriate activation of these signaling pathways is implicated in autoimmune conditions. Here, we report that indomethacin, a widely used nonsteroidal anti-inflammatory drug, inhibits nucleic acid-triggered IFN production. We found that both DNA- and RNA-stimulated IFN expression can be effectively blocked by indomethacin. Interestingly, indomethacin also prohibits the nuclear translocation of IRF3 following cytosolic nucleic acid recognition. Importantly, in cell lines and a mouse model of Aicardi–Goutières syndrome, indomethacin administration blunts self-DNA-induced autoimmune responses. Thus, our study reveals a previously unknown function of indomethacin and provides a potential treatment for cytosolic nucleic acid-stimulated autoimmunity.