The mechanisms involved in endothelial dysfunction are multifactorial. A correlation between oxidative stress and derangements of nitric oxide synthase (NOS) pathways in altered endothelial homeostasis has been most studied and demonstrated in different pathophysiological conditions. NOS activities are regulated by endogenous inhibitors such as asymmetric dimethyl-L-arginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Since recent data demonstrated that some endothelial dysfunction may be related to reduced expression and/or activity of DDAH, the aim of the present research was to investigate the expression of DDAH-2 and NOS isoforms in high glucose-mediated oxidative stress. Endothelial cells were incubated with normal (7 mM) and high concentrations (33 mM) of D-glucose for 5 days; mannose (26 mM) plus D-glucose (7 mM) was used as osmotic control. Data obtained in the present study show that the exposure for 5 days to high glucose increases oxidative stress, reduces DDAH-2 and eNOS expression and increases iNOS expression. These results indicate that DDAH-2 and iNOS/eNOS dysregulation may play a key role in high glucose-mediated oxidative stress, suggesting that selective modulation of DDAH isoforms may result in selective inhibition/activation of NOS isoforms, thereby providing a novel strategy of approach in vascular complications of several pathologies.