G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein-independent 5-HT(4) receptor (5-HT(4)R)-operated Src/ERK (extracellular signal-regulated kinase) pathway, but not the G(s) pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor' C-terminus in both human embryonic kidney (HEK)-293 cells and colliculi neurons. This inhibition required two sequences of events: the association of beta-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-t domain and the phosphorylation, by GRK5, of beta-arrestin1 (at Ser(412)) bound to the receptor. Phosphorylated beta-arrestin1 in turn prevented activation of Src constitutively bound to 5-HT(4)Rs, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that beta-arrestin1 phosphorylation by GRK5 regulates G protein-independent signalling.