Anemoside A3 (AA3) is a natural triterpenoid glycoside isolated from the root of Pulsatilla chinensis (Bunge) Regel. We previously showed that AA3 exhibits cognitive-enhancing and neuroprotective properties. In the present study, we demonstrated that AA3 modulates inflammatory responses by regulating prostaglandin E receptor 4 signaling. Because prostaglandin E receptor 4 is involved in the pathophysiology of experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), we assessed the beneficial effect of AA3 in EAE mice. AA3 treatment significantly reduced clinical severity and inflammatory infiltrates in the spinal cord of EAE mice. In vitro studies revealed that AA3 inhibited the T cell response toward the encephalitogenic epitope of myelin oligodendrocyte glycoprotein (MOG). AA3 significantly downregulated the expressions of certain Th1 and Th17 cytokines in activated T cells re-stimulated by MOG. Moreover, AA3 inhibited the activation of STAT4 and STAT3, which are the transcription factors pivotal for Th1 and Th17 lineage differentiation, respectively, in activated T cells. Pharmacological analysis further suggested that AA3 reduced Th17 cell differentiation and expansion. In conclusion, AA3 exerts an immunomodulatory effect in EAE, demonstrating its potential as a therapeutic agent for MS in humans.