<p>Multiple sclerosis (MS) is a chronic illness characterised by progressive central nervous system (CNS) degeneration. The efficacy of most current MS medicines is limited, and significant adverse effects remain a concern. STAT3 inhibitors show potential for treating several immunological diseases. The current study demonstrates that S3I-201, a well-established STAT3 inhibitor, effectively treats PLP_139−151-induced EAE in SJL/J mice. After establishing EAE, mice were administered S3I-201 intraperitoneally (10&#xa0;mg/kg) daily from day 14 through day 35. Flow cytometry was used to measure the effect of S3I-201 on the expression levels of IL-6, iNOS, Notch1, Notch3, GM-CSF, and NF-κB p65 in CD19⁺ and CD45R/B220⁺ B cells. RT-PCR was also used to study the effect of S3I-201 on the mRNA expression levels of IL-6, iNOS, Notch1, Notch3, GM-CSF, and NF-κB p65 in brain tissue. S3I-201 administration reduced the number of CD19⁺IL-6⁺, CD45R⁺IL-6⁺, CD19⁺iNOS⁺, CD45R⁺iNOS⁺, CD19⁺Notch1⁺, CD45R⁺Notch3⁺, CD19⁺GM-CSF⁺, CD45R⁺GM-CSF⁺, CD19⁺NF-κB p65⁺, and CD45R⁺NF-κB p65⁺ cells in EAE mice. Furthermore, S3I-201 administration reduced the expression of IL-6, iNOS, Notch1, Notch3, GM-CSF, and NF-κB p65 mRNA in brain tissue. S3I-201 inhibits the signalling of inflammatory mediators via various cellular pathways. Our findings suggest that S3I-201 may have therapeutic effects on EAE and may slow its progression in MS.</p>