We investigated the effect of tacrolimus, a calcineurin inhibitor, on dextran sulfate sodium (DSS)-induced colitis. After inducing colitis in C57BL/6 mice by administering DSS solution as drinking water for 7 d, the animals were treated with tacrolimus. Severity of colonic inflammation was evaluated based on colon weight per unit length. Levels of cytokines (interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-12, and tumor necrosis factor (TNF)-α) released from isolated inflamed colons of mice treated with tacrolimus or vehicle were also measured. Treatment with tacrolimus for 14 d reduced the colon weight per unit length and suppressed the release of IFN-γ and IL-1β, but not other cytokines, in inflamed colons of colitic mice compared with vehicle-treated mice. A positive correlation was noted between colon weight per unit length and released level of IFN-γ or IL-1β. The release of IFN-γ and IL-1β was also suppressed after single dosing with tacrolimus to colitic mice. Taken together, these results suggested that tacrolimus ameliorated DSS-induced colitis by suppressing release of IFN-γ and IL-1β from inflamed colon.