The peptide hormone oxytocin modulates socioemotional behaviour and sexual reproduction via the centrally expressed oxytocin receptor (OTR) across several species. Here, we report the crystal structure of human OTR in complex with retosiban, a non-peptide antagonist developed as an oral drug for the prevention of preterm labour. Our structure reveals insights into the detailed interactions between the G-protein coupled receptor (GPCR) and an OTR-selective antagonist. The observation of an extrahelical cholesterol molecule, binding in an unexpected location between helix IV and V, provides a structural rationale for its allosteric effect and critical influence on OTR function. Furthermore, our structure in combination with experimental data allows the identification of a conserved neurohypophyseal receptor-specific coordination site for Mg 2+ that acts as potent positive allosteric modulator for agonist binding.Together these results further our molecular understanding of the oxytocin/vasopressin receptor family and will facilitate structure-guided development of new therapeutics.[146 words]

The small cyclic neuropeptide hormone oxytocin (OT) and its cognate receptor play a central role in the regulation of social behaviour and sexual reproduction. Here we report the single-particle cryo-electron microscopy structure of the active oxytocin receptor (OTR) in complex with its cognate ligand oxytocin. Our structure provides high-resolution insights into the OT binding mode, the OTR activation mechanism as well as the subtype specificity within the oxytocin/vasopressin receptor family.