Background: Imidazoline compounds are well accepted to exhibit various pharmacological effects including antidepressant, anti-inflammatory, analgesic, anti-morphine tolerance and inhibit the activity of monoamine oxidase. 2-(-2-benzofuranyl)-2-imidazoline (2-BFI), a selective imidazoline 2 receptor (I2R) ligand, has been proven to exhibit therapeutic effects for various neuroimmunological diseases. However, the mechanism behind its neuroprotective properties remains elusive. Methods: In this study, we used 2-BFI for the treatment of mice with experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG33-55). The clinical signs of neurological deficits were evaluated daily. The demyelination and inflammatory infiltration in the CNS of mice with EAE were examined by Luxol Fast Blue (LFB) staining and hematoxylin-eosin (H&E) staining. Flow cytometry was utilized to examine the ratios of lymphocyte subsets in the periphery and CNS of mice with EAE. We also used Reverse Transcription–Polymerase Chain Reaction (RT-PCR) to observe the changes of expression of inflammatory cytokines by 2-BFI intervention. Results: We found that 2-BFI significantly reduced the incidence of EAE and attenuated the severity of neurological disability. Pathological staining showed that the infiltration of inflammatory cells and demyelination in the central nervous system (CNS) of the mice were markedly alleviated via 2-BFI intervention. To explore the mechanism of action of 2-BFI, we used flow cytometry to determine immunophenotypes in the spleen and CNS of the mice. We discovered that 2-BFI significantly decreased the ratio of CD28+ lymphocytes and B cells in the spleen of EAE mice. In the CNS, the expression of CD4+ T cells was downregulated by 2-BFI, while B cells and CD39+ lymphocytes were dramatically increased. RT-PCR also demonstrated that the level of IFN-γ mRNA secreted by CD4+T cells was lower than that in the CNS of EAE mice, while the levels of TGF- β and IL-10mRNA secreted by Treg and B cells were increased with 2-BFI intervention. Conclusion: 2-BFI could ameliorate EAE-induced neurobehavioral deficits and reduce the infiltration of inflammatory cells via regulating the activation and migration of lymphocyte subsets. This study provides a new explanation for the protective mechanism of 2-BFI in neuroimmune diseases.