IFI16, a PYHIN protein, is identified as an intracellular DNA sensor that mediates the induction of interferon-β (IFN-β). IFI16 directly binds to viral DNA motifs that stimulate IFN-β production and recruits STING, a critical mediator of IFN-β responses to DNA. Knockdown of IFI16 or its mouse ortholog p204 inhibits gene induction and activation of transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 is the first PYHIN protein shown to be involved in IFN-β induction, forming a new family of innate DNA sensors called 'AIM2-like receptors' (ALRs) with AIM2. These findings reveal a critical role for IFI16 in the detection of intracellular DNA and the induction of IFN-β, which is essential for antiviral responses.IFI16, a PYHIN protein, is identified as an intracellular DNA sensor that mediates the induction of interferon-β (IFN-β). IFI16 directly binds to viral DNA motifs that stimulate IFN-β production and recruits STING, a critical mediator of IFN-β responses to DNA. Knockdown of IFI16 or its mouse ortholog p204 inhibits gene induction and activation of transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 is the first PYHIN protein shown to be involved in IFN-β induction, forming a new family of innate DNA sensors called 'AIM2-like receptors' (ALRs) with AIM2. These findings reveal a critical role for IFI16 in the detection of intracellular DNA and the induction of IFN-β, which is essential for antiviral responses.