Hypoxia and hypoxia-inducible factors (HIF-1a and HIF-2a) modulate innate immune responses in the setting of systemic inflammatory responses and sepsis. The HIF-prolyl hydroxylase enzymes, PHD1, PHD2 and PHD3 regulate the mammalian adaptive response to hypoxia. However, their significance in the innate immune response is poorly understood. We found that genetic loss of PHD3 specifically shortens the survival of mice subjected to abdominal sepsis. In vivo, plasma levels of pro-inflammatory cytokines were enhanced, and recruitment of macrophages to internal organs was increased in septic PHD3-deficient mice, altogether indicating enhanced innate immune functions. Reciprocal bone marrow transplantation in sublethally irradiated mice revealed that the enhanced susceptibility of PHD3-deficient mice to sepsis-related lethality was specifically caused by loss of PHD3 in myeloid cells. In vitro assays revealed enhanced cytokine-production, migration, phagocytic capacity, and pro-inflammatory activation of PHD3-deficient macrophages. Increased pro-inflammatory activity of PHD3-deficient macrophages occurred concomitantly with enhanced HIF-1a protein stabilization and increased NF-kB activity, and interference with the expression of HIF-1a or NF-kB blunted their pro-inflammatory phenotype. It is concluded that loss or impairment of PHD3 enzyme function aggravates the innate immune response to septic stimuli via enhanced macrophage pro-inflammatory activity.