T effector memory (TEM) cells have a critical role in the secondary immune response and in the pathogenesis of different autoimmune diseases. Following activation, the number of Kv1.3 channels on the TEM cell membrane dramatically increases. Blockade of Kv1.3 channels results in inhibition of Ca2+ signaling in TEM cells, thus exerting an immunomodulatory effect. Since we observed that the peptide toxin (Vm24) isolated from the Mexican scorpion Vaejovis mexicanus completely and selectively blocked Kv1.3 channels currents, without impairing TEM cell viability, we decided to use it to investigate the molecular events that follow Kv1.3 blockade in human CD4+ TEM lymphocytes. We found that under TCR stimulation, Vm24 inhibited the expression of the activation markers CD25 and CD40L (but not that of CD69), the secretion of the pro-inflammatory cytokines IFN-γ, GM-CSF and TNF, as well as the release of the Th2 cytokines IL-4, IL-5, IL-9, IL-10, and IL-13. A similar inhibitory pattern was exerted by Vm24 on T cells isolated from patients with rheumatoid arthritis. On the other hand, a proteomic analysis of TCR-activated TEM cells indicated that the biological processes mainly affected by the blockade of Kv1.3 channels were cytokine-cytokine receptor interactions, mRNA processing via spliceosome, the response to unfolded proteins and intracellular vesicle transport, targeting the cell protein synthesis machinery. Altogether, these results underscore the role of Kv1.3 channels in regulating TEM lymphocyte function and highlight the potential use of the Vm24 peptide as an immunomodulatory agent for the therapy of conditions mediated by Th1 and Th2 lymphocytes.