As a member of the seven-transmembrane rhodopsin-like G protein-coupled receptor superfamily, the melanocortin-3 receptor (MC3R) is vital for the regulation of energy homeostasis and rhythms synchronizing in mammals, and its pharmacological effect could be directly influenced by the presence of melanocortin receptor accessory proteins (MRAPs), MRAP1 and MRAP2. The tetrapod amphibian Xenopus laevis (xl) retains higher duplicated genome than extant teleosts and serves as an ideal model system for embryonic development and physiological studies. However, the melanocortin system of the Xenopus laevis has not yet been thoroughly evaluated. In this work, we performed sequence alignment, phylogenetic tree, and synteny analysis of two xlMC3Rs. Co-immunoprecipitation and immunofluorescence assay further confirmed the co-localization and in vitro interaction of xlMC3Rs with xlMRAPs on the plasma membrane. Our results demonstrated that xlMRAP2.L/S could improve α-MSH-stimulated xlMC3Rs signaling and suppress their surface expression. Moreover, xlMC3R.L showed a similar profile on the ligands and surface expression in the presence of xlMRAP1.L. Overall, the distinct pharmacological modulation of xlMC3R.L and xlMC3R.S by dual MRAP2 proteins elucidated the functional consistency of melanocortin system during genomic duplication of tetrapod vertebrates.