Fast purinergic signaling is mediated by ATP and ATP-gated ionotropic P2X receptors (P2XRs), and it is implicated in pain-related behaviors. The properties exhibited by P2XRs vary between those expressed in heterologous cells and in vivo. Several modulators of ligand-gated ion channels have recently been identified, suggesting that there are P2XR functional modulators in vivo. Here, we establish a genome-wide open reading frame (ORF) collection and perform functional screening to identify modulators of P2XR activity. We identify TMEM163, which specifically modulates the channel properties and pharmacology of P2XRs. We also find that TMEM163 is required for full function of the neuronal P2XR and a pain-related ATP-evoked behavior. These results establish TMEM163 as a critical modulator of P2XRs in vivo and a potential target for the discovery of drugs for treating pain. Display omitted •Genome-wide ORF-based functional screening identifies a P2X receptor modulator•TMEM163 modulates P2X receptor channel properties and pharmacology•Neuronal P2X receptor function requires TMEM163•TMEM163 regulates ATP-evoked behavior Fast purinergic signaling is mediated by ATP-gated P2X receptors. Salm et al. perform functional screening with a genome-wide ORF collection and identify TMEM163, which modulates the channel properties and pharmacology of P2XRs and ATP-evoked behavior. They propose TMEM163 as a critical modulator of fast purinergic signaling.