The Smoothened receptor (SMO) mediates signal transduction in the hedgehog pathway, which is implicated in normal development and carcinogenesis. SMO antagonists can suppress the growth of some tumours; however, mutations at SMO have been found to abolish their antitumour effects, a phenomenon known as chemoresistance. Here we report three crystal structures of human SMO bound to the antagonists SANT1 and Anta XV, and the agonist, SAG1.5, at 2.6-2.8 Å resolution. The long and narrow cavity in the transmembrane domain of SMO harbours multiple ligand binding sites, where SANT1 binds at a deeper site as compared with other ligands. Distinct interactions at D473(6.54f) elucidated the structural basis for the differential effects of chemoresistance mutations on SMO antagonists. The agonist SAG1.5 induces a conformational rearrangement of the binding pocket residues, which could contribute to SMO activation. Collectively, these studies reveal the structural basis for the modulation of SMO by small molecules.