The innate immune response is the first line of defense against infection and relies on the ability of immune cells to detect the presence of infection through germline-encoded pattern recognition receptors. These include the Toll-like receptors, the retinoic acid inducible gene-like receptors, the nucleotide oligomerization domain-like receptors, and a number of DNA-sensing molecules. Members of the PYHIN protein family have recently emerged as sensors of microbial DNA. PYHIN proteins bind microbial DNA and form caspase-1-activating inflammasomes (AIM2) or drive type I IFN gene transcription (IFI16). Here, we review these discoveries and highlight the emerging role of the PYHIN protein family in mammalian host defenses.