The voltage-gated Kv1.3 K(+) channel in effector memory T cells serves as a new therapeutic target for multiple sclerosis. In our previous studies, the novel peptide ADWX-1 was designed and synthesized as a specific Kv1.3 blocker. However, it is unclear if and how ADWX-1 alleviates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. In this study, the administration of ADWX-1 significantly ameliorated the rat experimental autoimmune encephalomyelitis model by selectively inhibiting CD4(+)CCR7(-) phenotype effector memory T cell activation. In contrast, the Kv1.3-specific peptide had little effect on CD4(+)CCR7(+) cells, thereby limiting side effects. Furthermore, we determined that ADWX-1 is involved in the regulation of NF-κB signaling through upstream protein kinase C-θ (PKCθ) in the IL-2 pathway of CD4(+)CCR7(-) cells. The elevated expression of Kv1.3 mRNA and protein in activated CD4(+)CCR7(-) cells was reduced by ADWX-1 engagement; however, an apparent alteration in CD4(+)CCR7(+) cells was not observed. Moreover, the selective regulation of the Kv1.3 channel gene expression pattern by ADWX-1 provided a further and sustained inhibition of the CD4(+)CCR7(-) phenotype, which depends on the activity of Kv1.3 to modulate its activation signal. In addition, ADWX-1 mediated the activation of differentiated Th17 cells through the CCR7(-) phenotype. The efficacy of ADWX-1 is supported by multiple functions, which are based on a Kv1.3(high) CD4(+)CCR7(-) T cell selectivity through two different pathways, including the classic channel activity-associated IL-2 pathway and the new Kv1.3 channel gene expression pathway.