In a previous work, we showed an increased cell motility due to the accumulation and transcriptional activation of the Hypoxia Inducible Factor-1α (HIF-1α) and a reduced mitochondrial energy production in an in vitro model of endothelial dysfunction (ED) represented by human endothelial cells (ECs) chronically deprived of nitric oxide (NO) by L-NAME treatment. In the present study, in the attempt to unravel the pathway(s) linking NO deficiency to HIF-1α accumulation and activation, we focused our attention on Reactive Oxygen Species (ROS). We found that ROS were partially involved in HIF-1α stabilization, but not in the pro-migratory phenotype. Regarding mitochondrial dysfunction, it did not require neither ROS generation nor HIF-1α activity, and was not due to autophagy. Very interestingly, while acute treatment with L-NAME induced a transient increase in ROS formation, chronic NO deprivation by long term L-NAME exposure drastically reduced cellular ROS content giving rise to an antioxidant environment characterized by an increase in superoxide dismutase-2 (SOD-2) expression and activity, and by nuclear accumulation of the transcription factor NF-E2-related factor-2 (Nrf2). These results might have important implications for our understanding of the consequences of NO deprivation in endothelium behavior and in the onset of cardiovascular diseases.