There is evidence that the endothelial dysfunction would be caused by an increase of reactive oxygen species (ROS) and reactive nitrogen species (RNS) induced by high extracellular concentrations of D-glucose (González et al., 2011). In human fetal endothelium, the synthesis of ROS is mediated by NADPH oxidase (Villalobos et al., 2012); meanwhile the synthesis of nitric oxide (NO) is mediated by the L-arginine uptake dependent of human cationic amino acid transporter 1 (hCAT-1)(Shin et al., 2011). Our aim is describe the mechanisms underlie the effects of high extracellular D-glucose on synthesis of NO and ROS in human umbilical vein endothelial cells (HUVEC). Biological samples were obtained from normal pregnancies (ethics committee approval and informed patient consent were obtained). HUVEC were isolated by collagenase digestion (37 celcius degree) and cultured in medium 199 (M199) supplemented with 20% newborn and fetal calf sera. HUVEC were incubated (0-30 min or 1-24 h) with M199 containing 5 mM (control) or 25 mM of D-glucose (high D-glucose) en absence or presence of apocynin (100 µM, NADPH oxidase inhibitor), N-ethylmaleimide (NEM, 200 µM, CAT inhibitor), L-NAME (200 µM, eNOS inhibitor) or insulin (1 nM, 8 h). After incubations, were determined L-arginine uptake (100 µM L-arginine, 2 µCi/mL L-[3H]arginine), NO and ROS synthesis using diaminofluorescein (DAF-2DA) and dichlorofluorescein (DCF), respectively, and hCAT-1 and NADPH oxidase expression by real time PCR and western blot. High D-glucose increased (ANOVA unpaired Student’s t test, P<0.05, n=5-10) the L-arginine uptake after 10 (3±0.7-fold) and 30 (3±0.6-fold) min of incubation. These effects were blocked by pre-incubation with NEM and L-NAME. The acute incubation with high D-glucose not alter the ROS levels, but increased (P<0.05, n=3) the NO levels 2.1±04-fold and 2.8±0.6-fold after 10 and 30 min, respectively. Long term incubation with high D-glucose (24 h) increased (P<0.05, n=5-10) the L-arginine uptake (3±0.5-fold), ROS (1.8±0.1-fold) and NO (1.5±0.1-fold) levels. These effects of high D-glucose are related with increased (P<0.05, n=5-10) of mRNA (6±1-fold) and protein (2.7±0.2-fold) for hCAT-1 and mRNA for subunits of NADPH (1.5-fold). Insulin blocked the effects of high D-glucose on ROS, NO levels and hCAT-1 expression. In conclusion, the acute effect of high D-glucose is related with increases of L-arginine/NO pathway, without changes in ROS levels. Long term incubation with high D-glucose maintains the activation of this pathway but there is a concomitant activation of NADPH oxidase/ROS pathway. The endothelial dysfunction induced by high D-glucose could be prevented by insulin.