We examined the effect of bortezomib, a proteasome inhibitor, on the development of dextran sulfate sodium (DSS)-induced colitis in mice. DSS-colitis was induced by the administration of 3% DSS in water in C57BL/6J mice. Bortezomib was intraperitoneally administered daily for 9 days from the start of DSS. Ubiquitination of IκBα was evaluated by immunoblot. Bortezomib significantly ameliorated DSS-induced body weight loss and reduced the disease activity. The translocation of NF-κBp65 into the nucleus was markedly suppressed in the DSS + bortezomib group compared to the DSS group, but this difference was not detected in submucosal tissue. Ubiquitinated IκBα in the cytoplasm of colon epithelial cells was increased in the DSS + bortezomib group compared to the DSS group. In HT-29 cells, bortezomib blocked tumor necrosis factor-α (TNF-α)-induced nuclear translocation of NF-κB and this was accompanied by an increase in ubiquitinated IκBα in the cytoplasm. The mRNA expression of inflammatory mediators in colonic epithelial cells was significantly reduced by the treatment of bortezomib. Bortezomib inhibited the nuclear translocation of NF-κB in colonic epithelial cells by suppressing the degradation of IκBα and contributed to an improvement in DSS colitis. Our study suggests that bortezomib may be a new treatment option for IBD.