The trefoil factor (TFF) peptides are major secretory products of mucus cells of the gastrointestinal tract. There were evidences that administration of recombinant human TFF3 is effective in treatment of models of colitis, but the mechanism of the effects of rTFF3 is not fully understood. The main aims of this study is to evaluate effects of intraperitoneal injection recombinant human TFF3 on the expression of tumour necrosis factor alpha (TNF-alpha), toll-like receptor 4(TLR4), and nuclear factor kappaB (NF-kappaB) in trinitrobenzene sulphonic acid (TNBS) induced colitis mice. Distal colitis was induced in BALB/C mice by intracolonic administration of TNBS in ethanol. Treated with administration rhTFF3 for treatment group(5 mg/ml; approximately 0.5 mg/mouse), and normal saline for control for 5 consecutive days. Colonic damage score, tissue myeloperoxidase (MPO) activity, TLR4, NF-kappaB mRNA expression, and tissue TNF-alpha, TLR4, NF-kappaB production were determined, respectively. Once daily application of hTFF3 for 5 days after TNBS/ethanol had been injected, both microscopic and macroscopic injury and inflammatory index had been reduced compared with controls. In addition, decreased tissue TNF-alpha, TLR4, NF-kappaB production, and TLR4, NF-kappaB mRNA expression had been found. This study has shown that hTFF3 may have therapeutic potential in the treatment of inflammatory bowel disease, and one of the mechanisms may related to inhibit the TLR4/NF-kappaB signaling pathways.