IFI16 is an innate immune sensor for intracellular DNA. This study identifies IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-β (IFN-β). IFI16 directly binds to viral DNA motifs that induce IFN-β and recruits STING after DNA stimulation. Knockdown of IFI16 or its mouse ortholog p204 inhibits DNA-induced activation of IRF3 and NF-κB, as well as IFN-β induction. IFI16 is the first PYHIN protein shown to be involved in IFN-β induction. Together with AIM2, IFI16 forms a new family of innate DNA sensors called 'AIM2-like receptors' (ALRs). The innate immune response to DNA viruses involves the induction of IFN-β, which is mediated by STING, TBK1, and IRF3. While the role of DAI and RNA polymerase III in DNA sensing is known, their relevance in pathogen detection remains unclear. The study identifies IFI16 as a novel DNA sensor that activates the STING-TBK1-IRF3 pathway. IFI16 binds to viral DNA and recruits STING, leading to IFN-β induction. This process is independent of TLRs, DAI, and RNA polymerase III. The study also shows that IFI16 is required for DNA-mediated gene induction and signaling. Knockdown of IFI16 or p204 inhibits IFN-β induction in response to viral DNA. IFI16 is localized in the cytoplasm and binds to viral DNA, suggesting a direct role in DNA sensing. STING interacts with IFI16, and its recruitment is essential for IFN-β induction. The study demonstrates that IFI16 and p204 are critical for the innate immune response to DNA viruses, including HSV-1. The findings suggest that IFI16 and p204 are PYHIN proteins that act as sensors for exogenous DNA, but not RNA, leading to the activation of transcription factors and gene induction via a STING-dependent pathway. These proteins are essential for the innate immune response to DNA viruses and may also play a role in responses to bacterial pathogens and autoimmunity. The study highlights the importance of IFI16 and p204 in the innate immune response to DNA viruses and their potential role in antiviral immunity.IFI16 is an innate immune sensor for intracellular DNA. This study identifies IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-β (IFN-β). IFI16 directly binds to viral DNA motifs that induce IFN-β and recruits STING after DNA stimulation. Knockdown of IFI16 or its mouse ortholog p204 inhibits DNA-induced activation of IRF3 and NF-κB, as well as IFN-β induction. IFI16 is the first PYHIN protein shown to be involved in IFN-β induction. Together with AIM2, IFI16 forms a new family of innate DNA sensors called 'AIM2-like receptors' (ALRs). The innate immune response to DNA viruses involves the induction of IFN-β, which is mediated by STING, TBK1, and IRF3. While the role of DAI and RNA polymerase III in DNA sensing is known, their relevance in pathogen detection remains unclear. The study identifies IFI16 as a novel DNA sensor that activates the STING-TBK1-IRF3 pathway. IFI16 binds to viral DNA and recruits STING, leading to IFN-β induction. This process is independent of TLRs, DAI, and RNA polymerase III. The study also shows that IFI16 is required for DNA-mediated gene induction and signaling. Knockdown of IFI16 or p204 inhibits IFN-β induction in response to viral DNA. IFI16 is localized in the cytoplasm and binds to viral DNA, suggesting a direct role in DNA sensing. STING interacts with IFI16, and its recruitment is essential for IFN-β induction. The study demonstrates that IFI16 and p204 are critical for the innate immune response to DNA viruses, including HSV-1. The findings suggest that IFI16 and p204 are PYHIN proteins that act as sensors for exogenous DNA, but not RNA, leading to the activation of transcription factors and gene induction via a STING-dependent pathway. These proteins are essential for the innate immune response to DNA viruses and may also play a role in responses to bacterial pathogens and autoimmunity. The study highlights the importance of IFI16 and p204 in the innate immune response to DNA viruses and their potential role in antiviral immunity.