InSalmonellainfection, theSalmonellapathogenicity island-2 (SPI-2)-encoded type three secretion system (T3SS2) is of key importance for systemic disease and survival in host cells. For instance, in the streptomycin-pretreated mouse model SPI-2-dependentSalmonellareplication in lamina propria CD11c−CXCR1− monocytic phagocytes/macrophages (MΦ) is required for the development of colitis. In addition, containment of intracellularSalmonellain the gut critically depends on the antimicrobial effects of the phagocyte NADPH oxidase (PHOX), and possibly type 2 nitric oxide synthase (NOS2). For both antimicrobial enzyme complexes, oxygen is an essential substrate. However, the amount of available oxygen upon enteroinvasiveSalmonellainfection in the gut tissue and its impact onSalmonella–MΦ interactions was unknown. Therefore, we measured the gut tissue oxygen levels in a model ofSalmonellaenterocolitis using luminescence two-dimensionalin vivooxygen imaging. We found that gut tissue oxygen levels dropped from ˜78 Torr (˜11% O2) to values of ˜16 Torr (˜2% O2) during infection. Becausein vivovirulence ofSalmonelladepends on theSalmonellasurvival in MΦ,Salmonella–MΦ interaction was analysed under such low oxygen values. These experiments revealed an increased intracellular replication and survival of wild-type andt3ss2non-expressingSalmonella. These findings were paralleled by blunted nitric oxide and reactive oxygen species (ROS) production and reducedSalmonellaROS perception. In addition, hypoxia enhanced SPI-2 transcription and translocation of SPI-2-encoded virulence protein. Neither pharmacological blockade of PHOX and NOS2 nor impairment of T3SS2 virulence function alone mimicked the effect of hypoxia onSalmonellareplication under normoxic conditions. However, ift3ss2non-expressingSalmonellawere used, hypoxia did not further enhanceSalmonellarecovery in a PHOX and NOS2-deficient situation. Hence, these data suggest that hypoxia-induced impairment of antimicrobial activity andSalmonellavirulence cooperate to allow for enhancedSalmonellareplication in MΦ.