Hormone-producing enteroendocrine cells (EECs) are present throughout the gastrointestinal tract and respond to various nutrient and gut microbiota produced metabolites stimuli. Two important EEC subtypes, Glucagon like peptide-1 (GLP-1) producing L-cells and serotonin (5-HT) producing enterochromaffin (EC) cells interact via paracrine signaling and exhibit bidirectional regulation of expression and secretion of produced hormones. Accordingly, in vitro studies suggest potential to modulate 5-HT secretion by GLP-1 receptor agonism, and L-cell differentiation via serotonin receptor 4 agonism. However, the importance of this cellular signaling on host metabolism is poorly understood. In this study, we found that two weeks of high fat diet (HFD) feeding reduced RNA expression of gut hormones, including proglucagon (Gcg) gene encoding GLP-1 and Tryptophan hydroxylase1 (Tph1) gene encoding rate limiting enzyme in 5-HT synthesis, specifically in the colon and reduced plasma GLP-1 levels. Levels of propionate and butyrate were also reduced following HFD. However, supplementation of sodium propionate did not improve HFD induced reduction in GLP-1. In contrast, chemical induction of serotonin receptor 4 promoted GLP-1 levels, colonic Gcg RNA expression accompanied by improvement in glucose tolerance in HFD-fed mouse. Thus, this study suggests a novel mechanism to improve glucose tolerance via serotonin receptor 4 stimulation in the HFD induced obese mouse model.