Hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha display unique and sometimes opposing activities in regulating cellular energy homeostasis, cell fate decisions, and oncogenesis. Macrophages exposed to hypoxia accumulate both HIF-1alpha and HIF-2alpha, and overexpression of HIF-2alpha in tumor-associated macrophages (TAMs) is specifically correlated with high-grade human tumors and poor prognosis. However, the precise role of HIF-2alpha during macrophage-mediated inflammatory responses remains unclear. To fully characterize cellular hypoxic adaptations, distinct functions of HIF-1alpha versus HIF-2alpha must be elucidated. We demonstrate here that mice lacking HIF-2alpha in myeloid cells (Hif2aDelta/Delta mice) are resistant to lipopolysaccharide-induced endotoxemia and display a marked inability to mount inflammatory responses to cutaneous and peritoneal irritants. Furthermore, HIF-2alpha directly regulated proinflammatory cytokine/chemokine expression in macrophages activated in vitro. Hif2aDelta/Delta mice displayed reduced TAM infiltration in independent murine hepatocellular and colitis-associated colon carcinoma models, and this was associated with reduced tumor cell proliferation and progression. Notably, HIF-2alpha modulated macrophage migration by regulating the expression of the cytokine receptor M-CSFR and the chemokine receptor CXCR4, without altering intracellular ATP levels. Collectively, our data identify HIF-2alpha as an important regulator of innate immunity, suggesting it may be a useful therapeutic target for treating inflammatory disorders and cancer.