Elevated glucose concentrations have profound effects on cell function. We hypothesized that incubation of human aortic endothelial cells (HAEC) with high glucose increases insulin signaling and develops the appearance of insulin-stimulated glucose uptake by the cells. Compared with 5 mM glucose, incubation of HAEC with 30 mM glucose for up to 48 hours increased in a time-dependent manner expression of insulin receptor, IRS1, IRS2, and GLUT1 proteins. High glucose also increased the specific binding of ¹²⁵I-insulin in HAEC accompanied by accelerated production of IL-6 and IL-8. Short-term stimulation by 50 µU/ml insulin did not activate [¹⁴C] glucose uptake by HAEC incubated in 5 mM glucose. However, an addition of insulin to high glucose-exposed endothelial cells led to a significant increase in [¹⁴C] glucose uptake in a glucose concentration- and time-dependent fashion reaching a plateau at 48 hours of incubation. Furthermore, incubation of HAEC with 30 mM glucose resulted in a new insulin-stimulated ERK1,2 MAPK phosphorylation and increased lipid peroxidation and production of reactive oxygen species. These studies for the first time show that high glucose increases expression of insulin receptors and downstream elements of insulin signaling pathway and transforms "insulin-resistant" aortic endothelial cells into "insulin-sensitive" tissue regarding glucose uptake.