AbstractHistamine receptors are a group of G protein-coupled receptors (GPCRs) that play important roles in various physiological and pathophysiological conditions. Antihistamines that target the histamine H₁ receptor (H₁R) have been widely used to relieve the symptoms of allergy and inflammation. Here, to uncover the details of the regulation of H₁R by the known second-generation antihistamines, thereby providing clues for the rational design of newer antihistamines, we determine the cryo-EM structure of H₁R in the apo form and bound to different antihistamines. In addition to the deep hydrophobic cavity, we identify a secondary ligand-binding site in H₁R, which potentially may support the introduction of new derivative groups to generate newer antihistamines. Furthermore, these structures show that antihistamines exert inverse regulation by utilizing a shared phenyl group that inserts into the deep cavity and block the movement of the toggle switch residue W428^6.48. Together, these results enrich our understanding of GPCR modulation and facilitate the structure-based design of novel antihistamines.