Hypoxia is a potentially life-threatening stress condition of essential importance for endothelial cells. Moreover, endothelial cells play an important role in hypoxia-induced vascular disorders. The impact of hypoxia on the production of reactive oxygen species (ROS) is cell-specific. One of the most important sources of ROS in the vasculature is the NADPH Oxidase 4 (Nox4). We could previously show that Nox4 is the main Nox isoform in endothelial cells. However, the regulation of Nox4 under hypoxia in endothelial cells is unclear. First, we found a significant upregulation of Nox4 mRNA level after 24h of hypoxia (1% O₂) in human umbilical vein endothelial cells (HUVEC). In addition, levels of H₂O₂ produced by HUVEC were significantly elevated after hypoxia. Experiments using downregulation of Nox4 by lentiviral shRNA support an important role of Nox4 in this context. The response of the cells to hypoxia was confirmed by significant increase of the hypoxia-inducible factor (HIF) 1a expression on protein level. To further elucidate the cellular mechanisms behind this pattern of differential expression, we designed Nox4 promoter deletion constructs encompassing the first 1251 bp upstream from the transcription start site and the first 239 bp of exon 1. The -119/+239 Nox4 promoter construct is able to generate a significantly increased basal activity compared to the promoterless vector using dual-luciferase assay. Surprisingly, the longest and the shortest Nox4 promoter construct showed a comparable activity under hypoxic and normoxic conditions in highly transfectable human microvascular cells (HMEC-1), suggesting that the recently discovered hypoxia responsive element (HRE) in the Nox4 promoter (-391 to -387 bp) may be non-functional in endothelial cells. An upregulation of the VEGF promoter activity and of a Hypoxia Responsive Element (HRE3x) containing promoter have been used as positive controls. To further investigate this hypothesis, we overexpressed HIF-1a in HEK 293T cells and stabilized HIF-1a under normoxic conditions by treatment with DMOG (24h, 10 mM) in HUVEC. In addition, we blocked the HIF-pathway by adenoviral overexpression of a HIF-2a dominant-negative mutant in HUVEC. None of these experiments resulted in a significant change of Nox4 on the mRNA or promoter level, suggesting RNA stability of Nox4 might be affected under hypoxia. Taken together, these data improve our knowledge about the mechanisms of Nox4 regulation under hypoxia in endothelial cells and might support the development of novel therapeutic strategies for cardiovascular diseases.