Energy-sensing signals in the gut play a significant role in glucose metabolism, affecting hepatic glucose production and/or overall blood glucose levels; any imbalance of this system may cause diabetes. Recently, we reported that metformin activates energy sensor protein, AMP-protein kinase, in the upper small intestine to exert its anti-diabetic effect in rodents via a gut-brain axis. Although metformin action in the gut is subsequently reported to lower glucose levels in humans with type 2 diabetes as well, the overall intestinal mechanisms remain elusive. The mechanistic target of rapamycin (mTOR), a highly conserved energy-sensing kinase protein, is inhibited by metformin in vitro, and over-activation of mTOR in the liver and skeletal muscle leads to insulin resistance in rodents. The role of upper small intestinal mTOR in glucose homeostasis and/or metformin action is, however, unknown. Here, we first report that direct delivery of the mTOR inhibitor rapamycin or an adenovirus encoding the dominant negative-acting mTOR-mutated protein into the upper small intestine is sufficient to lower hepatic glucose production during the pancreatic (basal insulin)-euglycemic clamps in rodents with a high-fat diet (HFD)-induced insulin resistance. Second, we found that molecular (via upper small intestinal-targeted viral injection of adenovirus that expresses the constitutively active mTOR proteins) activation of upper intestinal mTOR blunted the glucose-lowering effect of the first-line anti-diabetic medication metformin. Finally, and most importantly, direct inhibition of upper small intestinal mTOR is sufficient to restore glycemia by lowering glucose production in type 2 diabetic rodents. In summary, our findings illustrate that inhibiting upper small intestinal mTOR is sufficient and necessary to lower glucose production and restore glucose homeostasis, and, thereby, unveil a previously unappreciated anti-diabetic role of mTOR in the upper small intestine.