Peripheral blood monocytes and macrophages are the only cell population with a proven hemoglobin (Hb) clearance capacity through the CD163 scavenger receptor pathway. Hb detoxification and related adaptive cellular responses are assumed to be essential processes to maintaining tissue homeostasis and promoting wound healing in injured tissues. Using a dual platform mass spectrometry analysis with MALDI-TOF/TOF and LTQ-Orbitrap instruments combined with isobaric tag for relative and absolute quantitation (iTRAQ), we analyzed how Hb exposure could modulate the macrophage phenotype on a proteome level. We identified and relatively quantified 3691 macrophage proteins, representing the largest human macrophage proteome published to date. The Hb polarized macrophage phenotype was characterized by an induced Hb:Hp-CD163-HO1-ferritin pathway and enhanced antioxidant enzymes while suppression of HLA class 2 was the most prominent effect. Enhanced Hb clearance and antioxidant capacity together with reduced antigen presentation might therefore be essential qualities of Hb polarized macrophages in wounded tissues and hemorrhage or atherosclerotic plaques.