D-glucose concentration in alveolar lining fluid is maintained much lower than that in the plasma, but is elevated by hyperglycemia. In the present study, we examined insulin uptake in A549 human alveolar epithelial cells; especially focusing on the effect of D-glucose on the uptake. Time- and temperature-dependence were observed in FITC-insulin uptake in A549 cells in the presence of D-glucose. Confocal laser scanning microscopic analysis showed the punctate localization of FITC-insulin in the cells. These results indicated that FITC-insulin was taken up by the cells through an endocytotic pathway. FITC-insulin uptake was increased 2–4 fold by extracellular D-glucose, but glucose analogs such as 3-O-methyl-D-glucose and 2-deoxy-D-glucose did not show any enhancement effect. Moreover, FITC-albumin and FD-4 uptake were not affected by D-glucose. Intracellular ATP amount was not affected by D-glucose during the incubation. Phloretin, a facilitative glucose transporter inhibitor, significantly inhibited D-glucose uptake as well as D-glucose-stimulated FITC-insulin uptake. Taken together, D-glucose may regulate the insulin uptake after being taken up by A549 cells, through the mechanism that is not directly related to ATP production. The effect of D-glucose on insulin absorption was also examined in in-vivo pulmonary administration study in rats, and co-administration of D-glucose potentiated the hypoglycemic action of insulin. These findings suggest that endocytosis is involved in insulin uptake, and D-glucose specifically enhances the endocytosis of insulin in alveolar epithelial cells. D-glucose concentration in alveolar lining fluid may play an important role in the absorption of insulin from the lung.