Signal transduction through G protein-coupled receptors (GPCRs) is regulated by receptor desensitization and internalization that follow agonist stimulation. Nitric oxide (NO) can influence these processes, but the cellular source of NO bioactivity and the effects of NO on GPCR-mediated signal transduction are incompletely understood. Here, we show in cells and mice that β-arrestin 2, a central element in GPCR trafficking, interacts with and is S-nitrosylated at a single cysteine by endothelial NO synthase (eNOS), and that S-nitrosylation of β-arrestin 2 is promoted by endogenous S-nitrosogluthathione. S-nitrosylation after agonist stimulation of the β-adrenergic receptor, a prototypical GPCR, dissociates eNOS from β-arrestin 2 and promotes binding of β-arrestin 2 to clathrin heavy chain/β-adaptin, thereby accelerating receptor internalization. The agonist- and NO-dependent shift in the affiliations of β-arrestin 2 is followed by denitrosylation. Thus, β-arrestin subserves the functional coupling of eNOS and GPCRs, and dynamic S-nitrosylation/denitrosylation of β-arrestin 2 regulates stimulus-induced GPCR trafficking.