It has been thought that μ-opioid receptors (MOPs) activate the G protein-mediated analgesic pathway and β-arrestin 2-mediated side effect pathway; however, ligands that only minimally recruit β-arrestin 2 to MOPs may also cause opioid side effects. Moreover, such side effects have been induced in mutant mice lacking β-arrestin 2 or expressing phosphorylation-deficient MOPs that do not recruit β-arrestin 2. These findings raise the critical question of whether β-arrestin 2 recruitment to MOP triggers side effects. Here, we show that β-arrestin 1 and 2 are essential in the efficient activation of the Gi/o-mediated MAPK signaling at MOP. Moreover, the magnitude of β-arrestin-mediated signals is not correlated with the magnitude of phosphorylation of the carboxyl-terminal of MOP, which is used to evaluate the β-arrestin bias of a ligand. Instead, the molecular association with β2-adaptin and clathrin heavy chain in the formation of clathrin-coated pits is essential for β-arrestin to activate MAPK signaling. Our findings provide insights into G protein-coupled receptor-mediated signaling and further highlight a concept that the accumulation of molecules required for endocytosis is critical for activating intracellular signaling.Copyright © 2022 Elsevier Inc. All rights reserved.