Experimental autoimmune encephalomyelitis (EAE) is the rodent model of multiple sclerosis (MS), a chronic autoimmune neuroinflammatory disease that has a devastating impact on various neurological functions of the patients. The hallmarks of both, MS and EAE, are neuroinflammation, demyelination and neuroaxonal degeneration. Various types of lymphoid and myeloid cells were shown to infiltrate the central nervous system and to participate in disease pathology. However, the role of eosinophil granulocytes has been less explored thus far. An early study showed that eosinophils infiltrate into the spinal cord of EAE mice and suggested their role in the disease progression. Recently, it was reported that eosinophils can play a protective role against EAE when mice are treated with an extract from helminths. Furthermore, it was shown that EAE development is not altered in mice deficient for interleukin-5, an important eosinophil pro-survival factor. Taken together, the role of eosinophils in EAE is currently unclear and needs to be investigated in detail. In the present study, we use the active model of EAE, whereby we immunized the C57BL/6 mouse strain with MOG35-55 peptide emulsified in the complete Freund’s adjuvant, in order to study a possible contribution of eosinophils to the disease pathology. Using the flow cytometry and RT-qPCR analysis of the spinal cord, we show that eosinophils infiltrate into the tissue in the course of EAE. The infiltration is likely driven by eosinophil chemoattractants, such as eotaxin-1, as the concentration of the latter was increased in the spinal cord during EAE, as shown on mRNA and protein level. Moreover, detailed flow cytometry analysis of spinal cord eosinophils revealed that they show signs of activation, namely an increase in CD11b and decrease in CCR-3 surface expression. Furthermore, we observed signs of degranulation of spinal cord eosinophils in EAE which was measured as a decrease of the side scatter parameter and an upregulation of CD63 surface expression. These data suggest a potential role of eosinophils in the pathology of EAE. In order to elucidate whether eosinophils are important for the disease development, eosinophil-deficient mice were subjected to EAE and the clinical development of the disease was observed. For this purpose, we used two independent models of eosinophil deficiency - ΔdblGATA1 and interleukin-5-depleted mice. ΔdblGATA1 mice are a genetically manipulated mouse strain bearing a deletion in GATA1 promoter that causes a specific depletion of eosinophils. Interestingly, clinical development of EAE was not affected in these mice when compared to their wild-type controls. As a next step, we depleted eosinophils by injecting wild-type mice with an antibody against the eosinophil pro-survival factor interleukin-5 in order to reduce eosinophil numbers in the effector phase of EAE. In accordance with the result from the experiment with ΔdblGATA1 mice, EAE progression was not altered in the eosinophil-depleted mice when compared to mice that were injected with an isotype control antibody. Further, we analyzed the neuroinflammation and demyelination in the spinal cord of 4ΔdblGATA1 mice subjected to EAE. Specifically, the infiltration of inflammatory cell populations, including CD4 and CD8 T cells, neutrophils and macrophages, was assessed by flow cytometry. In agreement with the unchanged clinical EAE development, inflammatory cell infiltration was not affected in ΔdblGATA1 mice. Furthermore, we analyzed expression of pro-inflammatory cytokines in the spinal cord of ΔdblGATA1 mice subjected to EAE in order to better characterize the inflammatory status. No significant changes were detected further confirming that eosinophils do not contribute to neuroinflammation in EAE. Finally, we assessed the demyelination in the spinal cord of ΔdblGATA1 EAE mice using luxol fast blue staining to detect myelin. In accordance with the unaffected clinical development and inflammatory status, we did not observe any difference in the spinal cord demyelination in ΔdblGATA1 mice when compared to their wild-type littermates. Taken together, although eosinophils infiltrate into the spinal cord of EAE mice and are activated and degranulate therein, they are dispensable for EAE development.