<jats:title>SUMMARY</jats:title><jats:p>In acute DSS-induced colitis nuclear factor (NF)-κB-dependent inflammatory cytokines including IL-1 and tumour necrosis factor-alpha (TNF-α) are up-regulated. Here we examined the effects of gliotoxin, a fungal metabolite known to inhibit NF-κB activity, on cytokine production by a mouse cell system in vitro and on intestinal inflammation and NF-κB activation in vivo. In vitro gliotoxin decreased TNF-α gene expression and protein production by RAW-264.7 mouse macrophage-like cells stimulated with lipopolysaccharide. In vivo, gliotoxin treatment of mice was begun on day 3 of 5% DSS application dissolved in the drinking water and continued until day 8. Gliotoxin treatment dose-dependently down-regulated colonic inflammation as assessed histologically and in parallel there was a suppression of colonic TNF-α and IL-1α mRNA expression on day 8 as analysed by semiquantitative reverse transcriptase-polymerase chain reaction (P &lt; 0·01). Furthermore, colonic NF-κB DNA-binding activity was increased in DSS-induced colitis and was suppressed by gliotoxin. These results demonstrate the essential role of NF-κB in DSS-induced colitis and indicate a molecular approach to the treatment of intestinal inflammatory disorders.