The human PYHIN proteins, AIM2, IFI16, IFIX, and MNDA, are regulators of immune response, transcription, apoptosis, and cell cycle. However, their protein interactions and underlying mechanisms remain largely uncharacterized. Here, we provide the interaction network for all PYHIN proteins. By designing a cell-based inducible system and integrating microscopy, immunoaffinity capture, quantitative mass spectrometry, and bioinformatics, we identified over 300 PYHIN interactions reflective of diverse functions.