The P2X1 receptor is under-researched, with limited structural and pharmacological data and a notable absence of drug-like antagonists. Genetic knockout studies in male mice showed 100% infertility, highlighting the P2X1 receptor’s promise as a target for non-hormonal male contraception.[1] Innovation in male contraception options is desperately needed, as over 300,000 unintended pregnancies occur daily and only two contraceptive options are currently available for men. Our aim is to to identify, characterise, and optimise small molecule antagonists of the P2X1 receptor as potential non-hormonal male contraceptives using integrated structural and pharmacological approaches. Novel P2X1 receptor ligands were identified using in silico and in vitro approaches. These, alongside known P2X1 receptor antagonists were assessed in a HEK293 cell line expressing P2X1 receptors using calcium mobilisation and radioligand binding assays. Cryogenic electron microscopy (cryo-EM) was used to determine the human P2X1 receptor structure in complex with NF449 and ATP. Cryo-EM structures revealed distinct ligand-binding modes at the P2X1 receptor. Newly identified P2X1 receptor ligands showed low micromolar potency and affinity, supporting their potential as drug leads. This work provides a platform to conduct further cryo-EM studies and use the resulting data to optimise new P2X1 receptor antagonists.