Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are regarded as 'incretins' working closely to regulate glucose homeostasis. Unimolecular dual and triple agonists of GLP-1R and GIPR have shown remarkable clinical benefits in treating type 2 diabetes. However, their pharmacological characterization is usually carried out in a single receptor-expressing system. In the present study we constructed a co-expression system of both GLP-1R and GIPR to study the signaling profiles elicited by mono, dual and triple agonists. We show that when the two receptors were co-expressed in HEK 293T cells with comparable receptor ratio to pancreatic cancer cells, GIP predominately induced cAMP accumulation while GLP-1 was biased towards β-arrestin 2 recruitment. The presence of GIPR negatively impacted GLP-1R-mediated cAMP and β-arrestin 2 responses. While sharing some common modulating features, dual agonists (peptide 19 and LY3298176) and a triple agonist displayed differentiated signaling profiles as well as negative impact on the heteromerization that may help interpret their superior clinical efficacies.