The heterotrimeric G protein-coupled serotonin receptor 5-HT receptor (5-HTR) mediates antinociception and may serve as a valuable target for the treatment of pain. Starting from a chemical library, we evolved ST171, a bitopic 5-HTR agonist that revealed highly potent and functionally selective G signaling without G activation and marginal β-arrestin recruitment. ST171 is effective in acute and chronic pain models. Cryo-electron microscopy structures of ST171 bound to 5-HTR in complex with the G protein compared to the canonical agonist befiradol bound to complexes of 5-HTR with G or G revealed that the ligands occupy different exo-sites. The individual binding poses are associated with ligand-specific receptor conformations that were further studied by molecular dynamics simulations, allowing us to better understand ligand bias, a phenomenon that may be crucial to the discovery of more effective and safe G protein-coupled receptor drugs.