Aims/hypothesis <p>Growing evidence implicates gut microbiota-derived metabolites in metabolic homeostasis. Indole, a microbial tryptophan metabolite, has been reported to enhance glucagon-like peptide-1 (GLP-1) secretion in vitro, and its derivatives have been inversely associated with risk of type 2 diabetes. We hypothesised that indole acts via the gastrointestinal tract to modulate glucose homeostasis, and tested this hypothesis using in vitro and in vivo models.</p> Methods <p>We measured GLP-1 secretion from cultured murine enteroendocrine cells, and evaluated intraperitoneal glucose tolerance and hormone secretion in mice following indole treatment. Subsequently, the impact of indole on intestinal epithelial cell fate and L&#xa0;cell number was examined using murine ileal organoid cultures and in vivo. Finally, we explored the effect of chronic indole administration on metabolic outcomes in a murine model of type 2 diabetes.</p> Results <p>Indole stimulated in vitro GLP-1 secretion in a concentration-dependent manner, and improved acute glucose management in vivo. Additionally, we demonstrate that indole drives enteroendocrine L&#xa0;cell differentiation in murine ileal organoids, resulting in increased L&#xa0;cell density and longer-term glucoregulatory benefits in vivo. Finally, sub-chronic indole administration improved glucose tolerance and insulin sensitivity in a diabetic mouse model.</p> Conclusions/interpretation <p>Our findings identify indole as a glucose-lowering molecule that acts on the gut, and raise the possibility of incorporating indole into nutraceutical supplements to aid in the treatment or prevention of type 2 diabetes. This study highlights the importance of gut microbiota-derived metabolites in metabolic health and opens new avenues for developing novel strategies to combat type 2 diabetes.</p> Data availability <p>RNA sequencing data are available from the Gene Expression Omnibus under accession number GSE306720.</p> Graphical Abstract <p></p>