Macrophages are major cells of the innate immunity. Macrophages derived from monocyte precursors undergo specific differentiation depending on the local tissue environment. Similar to the T helper type 1 and T helper type 2 polarization, two distinct states of polarized activation for macrophages have been defined in mouse and humans: the classically activated (M1) macrophage and the alternatively activated (M2) macrophage phenotypes. On the other hand, these different patterns of macrophage differentiation drive adaptive responses during the stages of infection, hence restraining inflammation and favoring tissue repair. In vitro generation and characterization of these subpopulations are essential to perform relevant studies understanding the host-pathogen interactions. Currently, several in vitro differentiation and polarization protocols are used to induce M1 or M2 mouse and human macrophages but none have been developed for the bovine species. We developed a method for in vitro differentiation and polarization of bovine macrophages using GM-CSF and IFNg to induce M1, and IL-4 to induce M2 phenotype. We characterized M1/M2 macrophages by specific morphology, production of cytokines (IL-10, IL-12, TNF-a), NO production, and phenotypic markers such as CD206 and TLR2.