Phosphorus is a macronutrient taken up by cells as inorganic phosphate (Pi). How cells sense cellular Pi levels is poorly characterized. Here, we report that SPX domains—which are found in eukaryotic phosphate transporters, signaling proteins, and inorganic polyphosphate polymerases—provide a basic binding surface for inositol polyphosphate signaling molecules (InsPs), the concentrations of which change in response toPiavailability. Substitutions of critical binding surface residues impair InsP binding in vitro, inorganic polyphosphate synthesis in yeast, and Pi transport inArabidopsis.In plants, InsPs trigger the association of SPX proteins with transcription factors to regulate Pi starvation responses. We propose that InsPs communicate cytosolic Pi levels to SPX domains and enable them to interact with a multitude of proteins to regulate Pi uptake, transport, and storage in fungi, plants, and animals.