Fasting is known to alter the function of various organs and the mechanisms of glucose metabolism, which affect health outcomes and slow aging. However, it remains unclear how fasting and feeding affects glucose absorption function in the small intestine. We studied the effects of the fasting and feeding on glucose-induced short-circuit current (Isc) in vitro using an Ussing chamber technique. Glucose-inducedIscby SGLT1 was observed in the ileum, but little or noIscwas observed in the jejunum in ad libitum-fed mice. However, in mice fasted for 24–48 h, in addition to the ileum, robust glucose-inducedIscwas observed over time in the jejunum. The expression of SGLT1 in the brush border membranes was significantly decreased in the jejunum under fed conditions compared to 48 h fasting, as analyzed by western blotting. Additionally, when mice were fed a 60% high glucose diet for 3 days, the increase in glucose-inducedIscwas observed only in the ileum, and totally suppressed in the jejunum. An increase in Na+permeability between epithelial cells was concomitantly observed in the jejunum of fasted mice. Transepithelial glucose flux was assessed using a non-metabolizable glucose analog,14C-methyl α-d-glucopyranoside glucose (MGP). Regardless of whether fed or fasted, no glucose diffusion mechanism was observed. Fasting increased the SGLT1-mediated MGP flux in the jejunum. In conclusion, segment-dependent up- and down-regulation mechanisms during fasting and feeding are important for efficient glucose absorption once the fast is broken. Additionally, these mechanisms may play a crucial role in the small intestine's ability to autoregulate glucose absorption, preventing acute hyperglycemia when large amounts of glucose are ingested.