Macrophages are innate immune cells which can react to a large number of environmental stimuli thanks to a high degree of plasticity. These cells are involved in a variety of tissue functions in homeostasis, and they play essential roles in pathological contexts. Macrophages’ activation state, which determines their functional orientation, is strongly influenced by the cellular environment. A large body of macrophage literature is devoted to better defining polarizations from a molecular viewpoint. It is now accepted that a multidimensional model of polarization is needed to grasp the broad phenotype repertoire controlled by environmental signals. The study presented here aimed, among other goals, to provide a molecular signature of various polarizations in human macrophages at the protein level so as to better define the different macrophage activation states. To study the proteome in human monocyte-derived macrophages as a function of their polarization state, we used a label-free quantification approach on in-gel fractionated and LysC/Trypsin digested proteins. In total, 5102 proteins were identified and quantified for all polarization states. New polarization-specific markers were identified and validated. Because oxygen tension is an important environmental parameter in tissues, we explored how environmental oxygen tension, at either atmospheric composition (18.6% O2) or “tissue normoxia” (3% O2), affected our classification of macrophage polarization. The comparative results revealed new polarization-specific makers which suggest that environmental oxygen levels should be taken into account when characterizing macrophage activation states. The proteomic screen revealed various polarization-specific proteins and oxygen sensors in human macrophages. One example is arachidonate 15-lipoxygenase (ALOX15), an IL4/IL13 polarization-specific protein, which was upregulated under low oxygen conditions and is associated with an increase in the rate of phagocytosis of apoptotic cells. These results illustrate the need to consider physicochemical parameters like oxygen level when studying macrophage polarization, so as to correctly assess their functions in tissue. .