Background Multiple sclerosis is a chronic demyelinating autoimmune disease accompanied by inflammation and loss of axons and neurons. Toll-like receptors play crucial roles in the innate immune system and inflammation. However, few studies have explored the specific effects of toll-like receptor 7 signaling pathway in multiple sclerosis. To explore underlying effects to develop a new therapeutic target, we use Vesatolimod, a safe and well-tolerated agonist of toll-like receptor 7, to assess the possible effects in Experimental autoimmune encephalomyelitis (EAE) animal model. Methods EAE animal model was induced by injection of MOG35-55 and monitored daily for clinical symptoms, and the treatment group was given Vesatolimod at the onset of illness. The therapeutic effects of Vesatolimod on EAE inflammation, demyelination, CD107b cells and T cells infiltration, and microglia activation was evaluated. Autophagy within the spinal cords of EAE mice was also preliminarily assessed. Results Treatment with Vesatolimod significantly alleviated clinical symptoms of EAE from day 18 post-immunization and decreased the expression levels of inflammatory cytokines, particularly Eotaxin and IL-12 (P40), in peripheral blood. It also inhibited demyelination in spinal cords. Moreover, VES treatment reduced activation of microglia, infiltration of CD3 + T cells and CD107b + cells, as well as inhibited the autophagy-related proteins expression in the spinal cords of EAE mice. Conclusion Our results indicate that Vesatolimod exhibits protective effects on EAE mice and is promising for treatment of MS.