The structural basis of ligand selectivity of G protein-coupled receptors for metabolic hormones has been an area of intense investigation, and yet it remains unresolved. One approach to delineating the mechanism of ligand-receptor interactions is to compare the ligand specificities of receptors expressed in species that emerged at different times within vertebrate evolution. In this paper we describe the isolation, functional, and phylogenetic characterization of the glucagon receptor from the goldfish Carassius auratus (Teleostei, order Cypriniformes), and compare its ligand specificity with that of the homologous rat receptor. Goldfish (gf) glucagon stimulated glucose production in a dose-dependent manner from isolated goldfish hepatocytes, resulting in 5-fold increase at 1 μm. The goldfish glucagon receptor (gfGlucR) shares 56, 51, 50, and 52% amino acid identities with frog Rana tigrina regulosa, mouse, rat, and human glucagon receptors, respectively. In competitive binding experiments, the recombinant gfGlucR displays high affinity toward goldfish, zebrafish, and human glucagons (IC50 = 0.6, 9, and 13 nm, respectively) but not toward goldfish glucagon-like peptide-1 or human glucagon-like peptide-1 (7-36) amide. Whereas both goldfish and human glucagons stimulated dose-dependent increases in intracellular cAMP through the recombinant gfGlucR, the recombinant rat GlucR interacted only with human glucagon, analogous to the specificity of the previously characterized glucagon receptor from the frog R. tigrina regulosa. Our results demonstrate that the binding pocket of gfGlucR can accommodate a broad range of glucagon structures and that in the frogs and mammals, there is a structural switch to a more restrictive conformation of glucagon receptors.