The Ca²⁺-activated K⁺ channel, K_Ca3.1 plays an important role in the regulation of T cell functions, and its blockades has been shown to decrease Ca²⁺ influx via Ca²⁺ release-activated Ca²⁺ channels in activated T cells. K_Ca3.1 is responsible for the pathogenesis of inflammatory bowel disease (IBD), and its blockade induced the down-regulation of the inflammatory cytokine, IFN-γ in Th1 cells and up-regulation of the anti-inflammatory cytokine, IL-10 in T_reg cells (Ohya et al., 2014 & 2021). The expression of IL-10 in T_reg cells is driven by various transcription factors (TFs) and upstream signaling pathways. We recently demonstrated that a K_Ca3.1 inhibitor up-regulated the expression of IL-10 in T_reg cells in the recovery phase in IBD model mice and in vitro-induced T_reg cells, together with the up-regulation of their TFs: Blimp1 and E4BP4. We here investigated the involvement of AP-1 (Fos/Jun) and NF-κB in the expression of IL-10 and its TFs in in vitro-induced mouse splenic T_reg cells. The pharmacological inhibition of JNK reversed K_Ca3.1 inhibition-induced increases in the expression of IL-10 and its TFs. The inhibition of K_Ca3.1 increased phosphorylated JNK and c-Jun levels. Therefore, the JNK/c-Jun signaling pathway may contribute to the K_Ca3.1 inhibition-induced up-regulation of IL-10 in peripherally-induced T_reg cells.