SignificanceAllosteric regulation, produced by the binding of a ligand at an allosteric site topographically distinct from the orthosteric site, represents a direct and efficient means for modulation of biological macromolecule function. Because allosteric modulators have advantages over classic orthosteric ligands as therapeutic agents, understanding the mechanism underlying allosteric modulation may open new therapeutic avenues. Here, we focused on allosteric regulation of P2X receptors, which are implicated in diverse pathophysiological processes, such as blood clotting, pain sensation, inflammation, and rheumatoid arthritis. Combining structural determination, molecular modeling, and mutagenesis, we identified a druggable allosteric site on P2X3. Our findings will facilitate the development of novel therapeutics targeting these receptors.