Glucagon-like peptide-1 receptor (GLP-1R) agonists are a highly effective class of therapies for type 2 diabetes (T2D), yet there are variable patient responses. Genomic variation in the human Glp1r altering structure, signal transduction and function, might be directly linked to therapeutic responses in patients. A naturally occurring low-frequency gain-of-function (GoF) missense variant, rs10305492 G>A (A316T), enriched in non-Finish Europeans, protects against T2D and cardiovascular disease. To examine the association between this mutation and glucose homeostasis, a knock-in mouse model bearing the homozygous A316T substitution in the human GLP-1R was made using CRISPR-Cas9 technology. These hGlp1rA316T/A316T mice displayed lower fasting blood glucose levels with improved glucose tolerance, plasma insulin, and insulin secretion, even under metabolic stress. They also exhibited alterations in islet cytoarchitecture and ³-cell identity, indicative of a compensatory mechanism under high-fat, high sucrose diet (HFHSD) challenge. Across all models investigated, the A316T variant exhibited characteristics of constitutive activation, including increased basal trafficking and signalling, with blunted incretin-induced responses which were more pronounced for certain GLP-1R agonists (GLP-1RA). This was further supported by structural insights from cryo-EM and computational analyses of the A316T compared to the WT GLP-1R. In conclusion, our study suggests that GLP-1R variants like the rs10305492 determine pharmacological responses to GLP-1R-targeting anti-diabetic therapies. This highlights the importance of precise molecular characterisation of GLP-1R genetic variants to predict individual responses to specific GLP-1R therapies and improved drug target validation techniques.