The intermediate-conductance Ca²⁺-activated K⁺- channel K_Ca3.1/KCNN4 plays an important role in the modulation of Ca²⁺ signaling through the control of the membrane potential in T-lymphocytes. Here, we study the involvement of K_Ca3.1 in the enlargement of the mesenteric lymph nodes (MLNs) in mouse model of inflammatory bowel disease (IBD). The mouse model of IBD was prepared by exposing male C57BL/6J mice to 5 % dextran sulfate sodium for 7 days. Inflammation-induced changes in K_Ca3.1 activity and the expressions of K_Ca3.1 and its regulators in MLN CD4⁺ T-lymphocytes were monitored by real-time PCR, Western blot, voltage-sensitive dye imaging, patch-clamp and flow cytometric analyses. Concomitant with an up-regulation of K_Ca3.1a and NDPK-B, a positive K_Ca3.1 regulator, an increase in K_Ca3.1 activity was observed in MLN CD4⁺ T-lymphocytes in the IBD model. Pharmacological blockade of K_Ca3.1 elicited the following results. 1) A significant decrease in IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation and crypt damage of the colon and MLN enlargement compared with control mice, and 2) the restoration of the expression levels of K_Ca3.1a, NDPK-B, and Th1 cytokines in IBD model MLN CD4⁺ T-lymphocytes. These findings suggest that the increase in K_Ca3.1 activity induced by the up-regulation of K_Ca3.1a and NDPK-B may be involved in the pathogenesis of IBD by mediating the enhancement of the proliferative response in MLN CD4⁺ T-lymphocyte and therefore, that the pharmacological blockade of K_Ca3.1 may decrease the risk of IBD.