The GPR15 receptor is a G protein-coupled receptor (GPCR), which is activated by an endogenous peptide GPR15L(25–81) and a C-terminal peptide fragment GPR15L(71–81). GPR15 signals through the G_i/o pathway to decrease intracellular cyclic adenosine 3′,5′-monophosphate (cAMP). However, the activation profiles of the GPR15 receptor within G_i/o subtypes have not been examined. Moreover, whether the receptor can also couple to G_s, G_q/11 and G_12/13 is unclear. Here, GPR15L(25–81) and GPR15L(71–81) are used as pharmacological tool compounds to delineate the GPR15 receptor-mediated Gα protein signalling using a G protein activation assay and second messenger assay conducted on living cells. The results show that the GPR15 receptor preferentially couples to G_i/o rather than other pathways in both assays. Within the G_i/o family, the GPR15 receptor activates all the subtypes (G_i1, G_i2, G_i3, G_oA, G_oB and G_z). The E_max and activation rates of G_i1, G_i2, G_i3, G_oA and G_oB are similar, whilst the E_max of G_z is smaller and the activation rate is significantly slower. The potencies of both peptides toward each G_i/o subtype have been determined. Furthermore, the GPR15 receptor signals through G_i/o to inhibit cAMP accumulation, which could be blocked by the application of the G_i/o inhibitor pertussis toxin.