Since their discovery, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET-release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia-inducible factor 1α (HIF-1α) by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21%) oxygen level and compared to hypoxic (1%) conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA) or Staphylococcus (S.) aureus wild-type and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was significantly increased under hypoxia. Interestingly, NET formation in response to viable S. aureus wild-type or nuclease-deficient strain was retained under hypoxia. Our results lead to the conclusion that hypoxia is not the ideal tool to analyze HIF-1α in neutrophils. However, the data clearly suggest that neutrophils react differently under hypoxia compared to normoxia and thereby highlight the importance of the usage of physiological relevant oxygen level when studying neutrophil functions.