The proteasome, a multicatalytic protease, is responsible for the degradation of intracellular proteins. Stimulation of cells with inflammatory cytokines, such as IFN-γ, leads to the replacement of the constitutive catalytic proteasome subunits by the inducible subunits low molecular mass polypeptide (LMP) 2 (β1i), multicatalytic endopeptidase complex-like-1 (β2i), and LMP7 (β5i), which are required for the production of certain MHC class I-restricted T cell epitopes. In this study, we investigated the effect of immunoproteasomes on the development of dextran sulfate sodium-induced colitis. Colitis induction in LMP2-, LMP7-, and multicatalytic endopeptidase complex-like-1–deficient mice caused reduced weight loss compared with wild-type mice. Although colon lengths were shortened in wild-type mice, no reduction was observed in immunoproteasome-deficient mice. In accordance with this, proinflammatory cytokines, such as TNF-α and IL-1β, were not upregulated in these mice. Blockage of LMP7 by a novel LMP7-selective inhibitor (PR-957) strongly reduced pathological symptoms of dextran sulfate sodium-induced colitis. Production of numerous cytokines in PR-957–treated mice was suppressed, resulting in reduced inflammation and tissue destruction. Taken together, these results demonstrate that an immunoproteasome-specific inhibitor can be used to attenuate autoimmune diseases like colitis.