The calcium-activated K⁺ channel KCa3.1 plays an important role in T lymphocyte Ca²⁺ signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca²⁺ influx. To assess the role of KCa3.1 channels in lymphocyte activation in vivo, we studied T cell function in KCa3.1^−/− mice. CD4 T helper (i.e., Th0) cells isolated from KCa3.1^−/− mice lacked KCa3.1 channel activity, which resulted in decreased T cell receptor–stimulated Ca²⁺ influx and IL-2 production. Although loss of KCa3.1 did not interfere with CD4 T cell differentiation, both Ca²⁺ influx and cytokine production were impaired in KCa3.1^−/− Th1 and Th2 CD4 T cells, whereas T-regulatory and Th17 function were normal. We found that inhibition of KCa3.1^−/− protected mice from developing severe colitis in two mouse models of inflammatory bowel disease, which were induced by (i) the adoptive transfer of mouse naïve CD4 T cells into rag2^−/− recipients and (ii) trinitrobenzene sulfonic acid. Pharmacologic inhibitors of KCa3.1 have already been shown to be safe in humans. Thus, if these preclinical studies continue to show efficacy, it may be possible to rapidly test whether KCa3.1 inhibitors are efficacious in patients with inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis.