Natural immunomodulator lactoferrin is known to exert an anti-inflammatory effect. However, there have been no studies that examine the mode of action of lactoferrin in reducing intestinal damage. We investigated the effect of lactoferrin on a trinitrobenzenesulfonic acid (TNBS)-induced colitis model in rats. Bovine lactoferrin was given once daily through gavage, starting 3 days before (preventive mode) or just after TNBS administration (treatment mode) until death. The distal colon was removed to be examined. Colitis was attenuated by lactoferrin via both modes in a dose-dependent manner, as reflected by improvement in macroscopic and histological scores and myeloperoxidase activity. Lactoferrin caused significant induction of the anti-inflammatory cytokines interleukin (IL)-4 and IL-10, significant reductions in the proinflammatory cytokines tumor necrosis factor- and IL-1, and downregulation of the nuclear factor-B pathway. We concluded that lactoferrin exerts a protective effect against colitis in rats via modulation of the immune system and correction of cytokine imbalance. Lactoferrin has potential as a new therapeutic agent for inflammatory bowel disease.