Background and Purpose: Although glucose absorption in the proximal small intestine and its regulatory mechanisms have been extensively studied, the regulation of glucose absorption in the distal small intestine is poorly understood. The present study investigates the mechanisms and regulation of glucose absorption in the ileum and compares with those in the jejunum. Experimental Approach: Glucose-induced short circuit current in isolated mouse intestinal epithelia was measured by the Ussing chamber. Key Results: Glucose induced a more pronounced short circuit current in the ileum than in the jejunum, exhibiting greater sensitivity to Na+ - glucose transporter inhibitors. The extracellular Ca2+ and endoplasmic reticulum Ca2+ storage in the ileum did not regulate glucose transport like the jejunum did. Blockers of Ca2+ and K+ channels did not significantly alter glucose-induced current in the ileum. Inhibition of Na+ - Ca2+ exchange and Na+ - H+ exchange reduced ileal glucose-induced current. Moreover, 5-HT reduced ileal glucose-induced current, which could be restored by selective inhibitors of 5-HT4R, adenyl cyclase and protein kinase A. Conclusion and Implications: The ileum has more pronounced glucose absorption, and its regulatory mechanisms significantly differ from those in the jejunum. Na+ - Ca2+ exchange and Na+ - H+ exchange regulate ileal glucose absorption, whereas 5-HT inhibits it via the 5-HT4R-cAMP- protein kinase A pathway.