The airway epithelium maintains differential glucose concentrations between the airway surface liquid (ASL, ~0.4 mM) and the blood/interstitium (5-6 mM), which is important for defense against infection. Glucose primarily moves from the blood to the ASL via paracellular movement, down its concentration gradient, across the tight junctions. However, there is evidence that glucose can move transcellularly across epithelial cells. Using a Förster resonance energy transfer sensor for glucose, we investigated intracellular glucose concentrations in airway epithelial cells and the role of hexokinases in regulating intracellular glucose concentrations in normoglycemic and hyperglycemic conditions. Our findings indicated that in airway epithelial cells (H441 or primary human bronchial epithelial cells) exposed to 5 mM glucose (normoglycemia), intracellular glucose concentration is in the micromolar range. Inhibition of facilitative glucose transporters (GLUTs) with cytochalasin B reduced intracellular glucose concentration. When cells were exposed to 15 mM glucose (hyperglycemia), intracellular glucose concentration was reduced. Airway cells expressed hexokinases I, II, and III. Inhibition with 3-bromopyruvate decreased hexokinase activity by 25% and elevated intracellular glucose concentration, but levels remained in the micromolar range. Exposure to hyperglycemia increased glycolysis, glycogen, and sorbitol. Thus, glucose enters the airway cell via GLUTs and is then rapidly processed by hexokinase-dependent and hexokinase-independent metabolic pathways to maintain low intracellular glucose concentrations. We propose that this prevents transcellular transport and aids the removal of glucose from the ASL and that the main route of entry for glucose into the ASL is via the paracellular pathway.