Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system characterized by motor deficits, cognitive impairment, fatigue, pain, and sensory and visual dysfunction. CD40, highly expressed in B cells, plays a significant role in MS pathogenesis. The experimental autoimmune encephalomyelitis (EAE) mouse model of MS has been well established, as well as its relevance in MS patients. This study aimed to evaluate the therapeutic potential of DAPTA, a selective C-C chemokine receptor 5 (CCR5) antagonist in the murine model of MS, and to expand the knowledge of its mechanism of action. Following the induction of EAE, DAPTA was administrated (0.01 mg/kg, i.p.) daily from day 14 to day 42. We investigated the effects of DAPTA on NF-κB p65, IκBα, Notch-1, Notch-3, GM-CSF, MCP-1, iNOS, and TNF-α in CD40+spleen B cells using flow cytometry. Furthermore, we also analyzed the effect of DAPTA on NF-κB p65, IκBα, Notch-1, Notch-3, GM-CSF, MCP-1, iNOS, and TNF-α mRNA expression levels using qRT-PCR in brain tissue. EAE mice treated with DAPTA showed substantial reductions in NF-κB p65, Notch-1, Notch-3, GM-CSF, MCP-1, iNOS, and TNF-α but an increase in the IκBα of CD40+B lymphocytes. Moreover, EAE mice treated with DAPTA displayed decreased NF-κB p65, Notch-1, Notch-3, GM-CSF, MCP-1, iNOS, and TNF-α and but showed increased IκBα mRNA expression levels. This study showed that DAPTA has significant neuroprotective potential in EAE via the downregulation of inflammatory mediators and NF-κB/Notch signaling. Collectively, DAPTA might have potential therapeutic targets for use in MS treatment.