Glucagon-like peptide 1 (GLP-1) is an incretin hormone secreted from enteroendocrine cells of the gut, that are called L cells. GLP-1 is released in response to the arrival of digested food in the gut, with glucose being one of the most potent stimulants of GLP-1 release. The mechanism of glucose-induced GLP-1 secretion has been well established. First glucose enters the L cells via a sodium-glucose co-transporter (SGLT1), the influx of sodium depolarises the cell membrane, which in turn opens the voltage-gated calcium channels and the subsequent rise in intracellular calcium levels triggers GLP-1 exocytosis. Previous reports showed that secretion of insulin from pancreatic β-cells, as well as glucagon from α-cells is pH sensitive. We therefore decided to investigate the GLP-1 secretion at different extracellular pH. Here, we report for the first time that secretion of GLP-1 from L-cells is also pH dependent.  We tested the effect of lowering the extracellular pH on GLP-1 secretion in two murine model L cells – GLUTag cells and primary intestinal cell cultures from wild-type mice. We discovered that lowering extracellular pH below neutral 7.0 decreases glucose-induced secretion of active GLP-1 by almost 50% at pH 6.3, while secretion remains unaltered at pH ranging 7.0-7.6.  We show that the effect of low extracellular pH on GLP-1 secretion can be mimicked by the inhibition of intracellular V-ATPase proton pumps by bafilomycin A1. This result strongly suggests that the pH dependence of GLP-1 secretion is mediated via proton influx and accumulation in the cytoplasm, rather than an external protonation effect.  We further speculated that the point of entry of protons might involve ion-coupled transport. We therefore replaced sodium in the secretion buffer with non-permeable cation, NMDG. As predicted, glucose-induced GLP-1 secretion was impaired by the absence of sodium and no pH dependence of GLP-1 secretion was observed. This would suggest the pH effect on GLP-1 secretion is Na+dependent. However, further depolarising the membrane with 30mM KCl, both in the presence or absence of sodium, still show a preserved pH dependence of GLP1 secretion, which would suggest that the pH effect is depolarision independent. Further study is required to investigate how the protons are imported into the cell as well as to elucidate the exact mechanism by which increased levels of intracellular protons decrease GLP-1 secretion. In a future study we will specifically address two pH sensitive steps involved in GLP-1 secretion: cleavage of proglucagon into active peptide by prohormone convertase 1 that would impact availability of the active GLP-1 for secretion and also docking/fusion of mature secretory vesicle into plasma membrane.  GLP-1 analogues and inhibitors of its degradation are currently under investigation as a new treatment of type 2 diabetes. Understanding how pH affects GLP-1 secretion could be of potential clinical relevance since intraluminal pH of the gut is very variable throughout and also changes with the food passage, health status of the patients and microflora content of the gut.