Nutrient absorption in the small intestine is essential for assimilation of nutrients required for energy and growth. To meet energy requirements, nutrients are efficiently absorbed from the luminal side against their chemical gradients. Many nutrients such as amino acids and glucose are actively absorbed across the brush border membrane of enterocytes via transporters that couple their movements to that of luminal Na+. Na+ coupling allows nutrients to be transported against their concentration from low luminal to higher intracellular concentration. It is therefore envisaged that Na+-dependent nutrient absorption mechanisms require a large amount of luminal Na+. However, little is known about how the intestine meets the needs of Na+ for nutrient absorption. It is thought that Na+ diffuses back into the lumen via paracellular pathways to support nutrient absorption. However, direct experimental evidence in support of this idea has not been shown. To investigate the relationship between paracellular Na+ movements and Na+-dependent glucose absorption, we took advantage of claudin 15 deficient (cldn15−/−) mice, which have been shown to have decreased paracellular Na+ permeability in the small intestine. Mice were anaesthetized with a mixture of drugs (10 µl/g, i.p.) consisting of medetomidine (30 µg/mL), midazolam (0.4 mg/mL) and butorphanol (0.5 mg/mL) and the small intestine was excised. The isolated segment was opened and mounted in an Ussing chambers. To investigate whether paracellular pathways are involved in Na+ recycling, we measured glucose-induced currents (ΔIsc) under open and short-circuit conditions and simultaneously measured changes in unidirectional 22Na+ fluxes (ΔJ). Under short-circuit conditions in wild-type mice, luminal application of glucose resulted in an increase in ΔIsc (13.7 ± 0.8 µmol/cm2/h) and an increase in unidirectional mucosal-to-serosal 22Na+ flux (ΔJNaMS, 12.5 ± 0.8 µmol/cm2/h) but not ΔJNaSM. However, under open-circuit conditions, equivalent glucose-induced ΔIsc (12.1 ± 0.8 µmol/cm2/h) was observed but ΔJNa(1.7 ± 1.3 µmol/cm2/h) was strongly inhibited. In the presence of phloridzin, both glucose-induced ΔIsc and ΔJ increments were totally abolished, suggesting that glucose-induced ΔJNaMS is mainly mediated by Na+-dependent glucose transporter SGLT1. Under short-circuit conditions, in cldn15−/− mice, luminal glucose induced an increase in ΔIsc (23.9 ± 3.8 µmol/cm2/h) and ΔJNaMS (21.4 ± 2.4 µmol/cm2/h) similar to that in wild-type mice. Unlike wild-type mice, though there was large negative luminal potential difference (Vte -20 mV), and a robust glucose-induced ΔJNaMS increment was observed (14.5 ± 1.9 µmol/cm2/h). These observations suggest that the Na+ which is absorbed by Na+-dependent glucose cotransport is rapidly recycled back into the lumen via paracellular pathways which are driven by increased luminal negativity.