The G protein-coupled glucagon-like peptide-1 receptor (GLP-1R) regulates glucose homeostasis by potentiating pancreatic insulin secretion, representing a key target for type 2 diabetes (T2D) treatment. Upon agonist-induced GLP-1R plasma membrane signalling, the GLP-1R enters the endocytic pathway where it is sorted to recycling and/or degradative compartments in a regulated manner. Since the endocytic pathway tightly regulates GPCR activity, understanding the mechanisms that control agonist-mediated GLP-1R trafficking might lead to identification of new T2D therapeutic targets and/or development of improved GLP-1R agonists. Herein, we have screened an siRNA library of GPCR trafficking regulators with the aim of identifying factors capable of controlling GLP-1R activity and pancreatic -cell responses. Based on their effect on exendin-4-induced insulin secretion, we have identified five factors [clathrin, dynamin1, AP2, and sorting nexins (SNXs) SNX27 and SNX1] that negatively regulate, and four [huntingtin-interacting protein 1 (HIP1), HIP14, G protein-coupled receptor associated sorting protein-1 (GASP-1), and Neural Precursor Cell Expressed, Developmentally Down-Regulated 4 (Nedd4)], that enhance GLP-1R activity. Amongst these, we selected three candidates (HIP1, SNX1 and SNX27) for further characterisation. HIP1 was found to modulate GLP-1R G protein coupling while concomitantly controlling GLP-1R internalisation, while the two SNXs were shown to control GLP-1R endosomal sorting, affecting GLP-1R recycling versus degradation rates and determining endosomal and cell surface responses. In parallel, we investigated the role of the plasma membrane lipid composition on GLP-1R signalling and trafficking. Upon ligand binding, GLP-1R was shown to undergo palmitoylation and partitioning into specific cholesterol-enriched plasma membrane nanodomains, leading to GLP-1R signal transduction and cholesterol-dependent internalisation. This process was controlled by specific agonist binding kinetics and positive allosteric modulation. We have therefore identified key endocytic factors that determine GLP-1R activity and -cell incretin responses. Furthermore, we have demonstrated that both GLP-1R activity and trafficking are modulated by interactions with the surrounding lipid bilayer.