Multiple sclerosis is considered to be initiated by a deregulated, myelin-specific T cell response. However, the formation of inflammatory CNS lesions and the contribution of different leukocyte subsets in setting up these lesions are still incompletely understood. In this study, we show that, in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis, neutrophil granulocytes are important contributors in preparing CNS inflammation. Preclinical single-dose Ab-mediated depletion of neutrophils delayed the onset and continuous depletion attenuated the development of experimental autoimmune encephalomyelitis, whereas the generation of a myelin-specific T cell response remained unaffected. Neutrophil-related enzymes such as myeloperoxidase and neutrophil elastase did not contribute in mounting CNS inflammation, as analyzed by using respective knockout mice and inhibitors. CNS-infiltrating neutrophils secreted proinflammatory molecules and matured bone marrow-derived dendritic cells in vitro, which in turn enhanced their ability to restimulate myelin-specific T cells. This was mirrored in vivo, in which depletion of neutrophils specifically impaired maturation of microglia and macrophages into professional APCs, resulting in a diminished amplification of early CNS inflammation. Therefore, inside the CNS neutrophils provide local cofactors that are required for the maturation of myeloid cells into professional APCs representing an essential step for the local restimulation of myelin-specific T cells and the development of autoimmune disease.