Acupuncture at ST36 can alleviate a variety of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), while the specific mechanism for the treatment of EAE is not clear. In this study, we found that acupuncture at ST36 can significantly increase the excitability of splenic sympathetic nerve, and promote the differentiation of peripheral B and CD4⁺T cells in the anti-inflammatory direction. After blocking the splenic sympathetic nerve with 6-OHDA, this anti-inflammatory effect of acupuncture is partially reversed. In addition, the results of western blot and qPCR showed that acupuncture at ST36 simultaneously activated the β2-AR-cAMP signaling pathway in the splenic B and CD4⁺T cells, and this activation was more significant in B cells. In vitro, when CD4⁺T cells were cultured alone, norepinephrine (NE) had no significant effect on their differentiation. While in the presence of B cells, NE significantly promotes the anti-inflammatory differentiation of B and CD4⁺T cells. Therefore, the above results reveal that acupuncture can relieve EAE by stimulating the sympathetic nerves of spleen, mainly through acting on B cells to mediate anti-inflammatory effects, and indirectly affecting the function of CD4⁺T cells.