The etiology of atherosclerosis that occurs in diabetes mellitus is unclear. Previously, adenosine has been shown to inhibit growth of rat aortic smooth muscle cells. Nucleoside transporters play an integral role in adenosine function by regulating adenosine levels in the vicinity of adenosine receptors. Therefore, we studied the effect of 25 mM D-glucose which mimics hyperglycemia of diabetes, on adenosine transport in cultured human aortic smooth muscle cells (HASMCs). Although RT-PCR demonstrated the presence of equilibrative nucleoside transporter-1 (ENT1) and ENT2 mRNA, functional studies revealed that adenosine transport in HASMCs was predominantly mediated by ENT1 and was inhibited by nitrobenzylmercaptopurine riboside (NBMPR) with an IC₅₀ of 0.69 ± 0.05 nM. Adenosine transport in HASMCs was increased by > 30% after treatment for 48 hrs with 25 mM D-glucose. Kinetic studies showed that D-glucose increased the V_max of adenosine transport without affecting the K_m. Similarly, D-glucose increased the B_max of high affinity [³H]NBMPR binding while the K_d was not changed. Consistent with these observations, 25 mM D-glucose increased both the mRNA and protein expression of ENT1. Treatment of serum-starved cells with the selective inhibitors of MAPK/ERK, PD98059 (40 µM) and U0126 (10 µM), abolished the effect of D-glucose on ENT1. We conclude that D-glucose up-regulates the protein and message expression and functional activity of ENT1 in HASMCs, possibly via MAPK/ERK dependent pathways. Pathologically, the increase in ENT1 activity in diabetes may affect the availability of adenosine in the vicinity of adenosine receptors and thus alters vascular functions in diabetes.