Mononuclear phagocytes are key regulators of both tissue damage and repair in neuroinflammatory conditions such as multiple sclerosis. To examine divergent phagocyte phenotypes in the inflamed CNS, we introduce an in vivo imaging approach that allows us to temporally and spatially resolve the evolution of phagocyte polarization in a murine model of multiple sclerosis. We show that the initial proinflammatory polarization of phagocytes is established after spinal cord entry and critically depends on the compartment they enter. Guided by signals from the CNS environment, individual phagocytes then switch their phenotype as lesions move from expansion to resolution. Our study thus provides a real-time analysis of the temporospatial determinants and regulatory principles of phagocyte specification in the inflamed CNS.