20-HETE increases the expression of Vascular Endothelial Growth Factor (VEGF) in human dermal microvascular endothelial cells (EC). VEGF is regulated by Hypoxia Inducible Factor (HIF-1). We studied whether 20-HETE also upregulates HIF-1 using the stable 20-HETE analog WIT003, (20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid, 1-10 µM), and found that it induced a marked increase in VEGF and HIF-1 protein levels. The increases in VEGF preceded the changes in HIF-1 and a VEGF neutralizing antibody prevented the increases in HIF-1. Stimulation with exogenously added VEGF also led to HIF-1 upregulation. This suggests that 20-HETE first causes increases in VEGF, which then causes the upregulation of HIF-1. Addition of WIT003 results in a rapid and sustained increase in superoxide formation. When WIT003 was added in the presence of the NOS inhibitor L-NAME, no changes in superoxide, VEGF, or HIF-1 were observed. This suggests that NOS is responsible for the early changes induced by WIT003, likely because of NOS-uncoupling. Furthermore, WIT003 induced the expression of the NADPH oxidase subunit p47^phox in EC prior to the increases in HIF-1. Incubation with PEG-SOD (400 U/ml), apocynin (100 µM), DPI (10 µM) or p47^phox down regulation with siRNA all inhibited the increases in HIF-1 expression. This indicates that the early changes in superoxide lead to VEGF increases and thereby NADPH oxidase-dependent superoxide production is required for the increases in HIF-1. Incubation with the MEK1/ERK1/2 inhibitor U0126 (10 µM) completely abolishes the increases in VEGF and thus HIF-1, suggesting involvement of ERK1/2 activation. We also found that the higher HIF-1 expression induced by WIT003 was accompanied by higher expression of erythropoietin receptor and angiopoietin-2 proteins. These increases were caused by HIF-1 since their levels were markedly decreased by downregulation of HIF-1 with siRNA. 20-HETE may be a novel non-hypoxic regulator of HIF-1, and HIF-1-regulated genes in EC.