G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent productive drug targets . Orphan GPCRs, which have unknown endogenous ligands, are considered drug targets and consequently have attracted great interest in identifying their endogenous cognate ligands for deorphanization . However, additional studies have shown that GPCRs, including many orphan GPCRs, can constitutively activate G protein signaling in a ligand-independent manner . GPR39 is such an orphan GPCR with constitutive activity . Here, we performed a phylogenetic and selection analysis of GPR39 in vertebrates, and we found that GPR39 underwent positive selection in different branches of vertebrates . Using luciferase reporter assays, we demonstrated that human, frog and chicken GPR39 can constitutively activate Gq and G12 signaling pathways in a ligand-independent manner . Zebrafish GPR39 can constitutively activate Gs, Gq and G12 signaling pathways in a ligand-independent manner . We further found that the zebrafish-H296 site is crucial for the activity of the Gs signaling pathway . In addition, our mutagenesis studies indicated that the positive selection sites of GPR39 from different species had important effects on the constitutive activity of the receptor . Our results revealed the adaptive evolution of GPR39 in diverse directions, which led to differences in constitutive activity.