The vasoactive peptide urotensin-II (U-II) has been implicated to activate vascular proliferative responses, although the underlying mechanisms are unclear. Reactive oxygen species (ROS) generated by NADPH oxidases have been identified as signalling molecules in the angiogenic response, whereas hypoxia-inducible factor-1 (HIF1) is a major regulator of proangiogenic gene expression under hypoxia. We thus hypothesized that U-II could promote angiogenesis involving NOX2 and HIF1a. U-II increased formation of capillaries in an in vitro matrigel assay, and this response was prevented by the urotensin-receptor antagonist urantide as well as by downregulation of HIF-1a. Conversely, U-II increased HIF transcriptional activity as well as HIF-1a protein levels. Interestingly, U-II was able to not only increase NOX2-dependent ROS generation, but also to induce NOX2 mRNA and protein expression in a HIF-1a dependent manner since overexpression of HIF1a increased NOX2 expression and ROS generation, whereas HIF1a depletion prevented these responses. In addition, chromatin immunoprecipitation indicated HIF1a binding to the NOX2 gene. Importantly, U-II-induced angiogenesis was dependent on NOX2 as demonstrated by in vitro matrigel assays as well as in a vivo angiogenesis assay in NOX2-/- mice. Taken together, these findings show that HIF1a promotes angiogenesis also under non-hypoxic conditions in response to U-II by upregulating the expression of the NADPH oxidase subunit NOX2. The identification of NOX2 as a target gene of HIF1 provides novel insights into the role of HIF1 and NOX2 in regulating angiogenesis and may provide the basis of new therapeutic strategies to combat diseases associated with disordered angiogenesis.