The gut microbiota alters energy homeostasis. In parallel, metformin regulates upper small intestinal sodium glucose cotransporter-1 (SGLT1), but whether changes of the microbiota or SGLT1-dependent pathways in the upper small intestine mediate metformin action is unknown. Here we report that upper small intestinal glucose sensing triggers an SGLT1-dependent pathway to lower glucose production in rodents. High-fat diet (HFD) feeding reduces glucose sensing and SGLT1 expression in the upper small intestine. Upper small intestinal metformin treatment restores SGLT1 expression and glucose sensing while shifting the upper small intestinal microbiota partly by increasing the abundance of Lactobacillus. Transplantation of upper small intestinal microbiota from metformin-treated HFD rats to the upper small intestine of untreated HFD rats also increases the upper small intestinal abundance of Lactobacillus and glucose sensing via an upregulation of SGLT1 expression. Thus, we demonstrate that metformin alters upper small intestinal microbiota and impacts a glucose-SGLT1-sensing glucoregulatory pathway. Display omitted •Upper small intestinal glucose sensing activates SGLT1 to lower glucose production•High-fat diet reduces glucose-SGLT1 sensing and decreases Lactobacillus•Metformin restores glucose-SGLT1 sensing while increasing Lactobacillus•Metformin-treated microbiota transplants restore glucose-SGLT1 sensing Bauer et al. identify a glucose-sensing pathway in the upper small intestine that lowers glucose production in rodents. A high-fat diet shifts the upper small intestinal microbiota and compromises glucose sensing, while metformin treatment in the upper small intestine counteracts the microbiota shift and restores glucose sensing.