OBJECTIVE: We aimed to assess the spatiotemporal GLP-1 and GIP receptor signaling, trafficking and recycling dynamics of GIPR mono-agonists, GLP-1R mono-agonists including semaglutide, and GLP-1/GIP dual-agonists MAR709 and tirzepatide. METHODS: Receptor G protein recruitment and internalization/trafficking dynamics were assessed using bioluminescence resonance energy transfer (BRET)-based technology and live cell HILO microscopy. RESULTS: Relative to the native and acylated GLP-1 agonists, MAR709 and tirzepatide show preserved maximal cAMP production despite partial Gαs recruitment paralleled by diminished ligand-induced receptor internalization at both target receptors. Despite MAR709's lower internalization rate, GLP-1R co-localization with Rab11-associated recycling endosomes was not different between MAR709 and GLP-1R specific mono-agonists. CONCLUSIONS: Our data indicate that MAR709 and tirzepatide induce unique spatiotemporal GLP-1 and GIP receptor signaling, trafficking and recycling dynamics relative to the native peptides, semaglutide and matched mono-agonist controls. These findings support the hypothesis that the structure of the GLP-1/GIP dual-agonists confer a biased agonism that, in addition to its influence on intracellular signaling, uniquely modulates receptor trafficking.