Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target. Display omitted •NLRC3 binds to herpes simplex virus dsDNA via the LRR domain with high affinity•DNA binding to NLRC3 diminishes its interaction with STING and TBK1 to enhance IFN-I•DNA binding to NLRC3 enhances ATPase activity•The ATPase activity of NLRC3 is crucial for DNA-mediated dissociation of STING NLRC3 is a negative regulator that attenuates type I interferon by sequestering STING and TBK1 activation, but the ligand of NLRC3 is unknown. Li et al. demonstrate that viral DNA directly binds to NLRC3 through the LRR domain, which enhances its ATPase activity and unleashes its interaction with STING and TBK1.