Abstract Background Multiple sclerosis (MS) is a chronic debilitating immune-mediated disease of the central nervous system (CNS) driven by demyelination and gray matter neurodegeneration. We previously reported an experimental autoimmune encephalomyelitis (EAE) MS mouse model with elevated serum CXCL1 that developed severe and prolonged neuron damage. Our findings suggested that CXCR2 signaling may be important in neuronal damage, thus implicating neutrophils, which express CXCR2 in abundance, as a potential cell type involved. The goals of this study were to determine if CXCR2 signaling in neutrophils mediate neuronal damage and to identify potential mechanisms of damage. Methods EAE was induced in wild-type control and neutrophil-specific Cxcr2 knockout (Cxcr2 cKO) mice by repeated high-dose injections of heat-killed Mycobacterium tuberculosis and MOG_35–55 peptide. Mice were examined daily for motor deficit. Serum CXCL1 level was determined at different time points throughout disease development. Neuronal morphology in Golgi-Cox stained lumbar spinal cord ventral horn was assessed using recently developed confocal reflection super-resolution technique. Immune cells from CNS and lymphoid organs were quantified by flow cytometry. CNS-derived neutrophils were co-cultured with neuronal crest cells and neuronal cell death was measured. Neutrophils isolated from lymphoid organs were examined for expression of reactive oxygen species (ROS) and ROS-related genes. Thioglycolate-activated neutrophils were isolated, treated with recombinant CXCL1, and measured for ROS production. Results Cxcr2 cKO mice had less severe disease symptoms at peak and late phase when compared to control mice with similar levels of CNS-infiltrating neutrophils and other immune cells despite high levels of circulating CXCL1. Additionally, Cxcr2 cKO mice had significantly reduced CNS neuronal damage in the ventral horn of the spinal cord. Neutrophils isolated from control EAE mice induced vast neuronal cell death in vitro when compared with neutrophils isolated from Cxcr2 cKO EAE mice. Neutrophils isolated from control EAE mice, but not Cxcr2 cKO mice, exhibited elevated ROS generation, in addition to heightened Ncf1 and Il1b transcription. Furthermore, recombinant CXCL1 was sufficient to significantly increase neutrophils ROS production. Conclusions CXCR2 signal in neutrophils is critical in triggering CNS neuronal damage via ROS generation, which leads to prolonged EAE disease. These findings emphasize that CXCR2 signaling in neutrophils may be a viable target for therapeutic intervention against CNS neuronal damage.