Metallothioneins (MTs) are a family of cysteine-rich low molecular-weight proteins that can act as reactive oxygen species scavengers. Although it is known that the induction of MT expression suppresses various inflammatory disorders, the role of MTs in intestinal inflammation remains unclear. In this study, we investigated the effects of dextran sulfate sodium (DSS) administration in mice with targeted deletions of the MT-I/II genes. Acute colitis was induced by 2% DSS in male MT-I/II double knockout (MT-null) and C57BL/6 (wild-type) mice. The disease activity index (DAI) was determined on a daily basis for each animal, and consisted of a calculated score based on changes in body weight, stool consistency and intestinal bleeding. Histology, colon length, myeloperoxidase (MPO) activity and colonic mRNA expression and the concentration of inflammatory cytokines were evaluated by real-time-PCR and enzyme-linked immunosorbent assay (ELISA). The localization of MTs and macrophages was determined by immunohistological and immunofluorescence staining. To investigate the role of MTs in macrophages, peritoneal macrophages were isolated and their responses to lipopolysaccharide were measured. Following DSS administration, the DAI score increased in a time-dependent manner and was significantly enhanced in the MT-I/II knockout mice. Colonic MPO activity levels and inflammatory cytokines [tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and interleukin (IL)-17] production increased following DSS administration, and these increases were significantly enhanced in the MT-I/II knockout mice compared with the wild-type mice. MT-positive cells were detected in the lamina propria and submucosal layer by immunohistochemical and immunofluorescence staining, and were mainly co-localized in F4/80-positive macrophages. The production of inflammatory cytokines (TNF-alpha, IFN-gamma and IL-17) from isolated peritoneal macrophages increased following lipopolysaccharide stimulation, and these increases were significantly enhanced in the macrophages obtained from the MT-I/II knockout mice. These data indicate that MTs play an important role in the prevention of colonic mucosal inflammation in a mouse model of DSS-induced colitis, thus suggesting that endogenous MTs play a protective role against intestinal inflammation.