Tumor necrosis factor-α (TNF-α) is a key cytokine involved in the pathogenesis of inflammatory bowel disease. We have developed a second-generation antisense oligonucleotide (ISIS 25302) specific for murine TNF-α and have evaluated this oligonucleotide in two models of gut inflammation of distinct etiology. ISIS 25302 decreased TNF-α mRNA in a dose- and sequence-dependent manner in vitro in the mouse macrophage cell line P388D1. It also reduced TNF-α mRNA in vivo, in whole adipose tissue and in macrophages isolated from the adipose tissue of db/db mice, a strain with constitutively high expression of TNF-α. ISIS 25302 significantly reduced disease activity index scores in mice with both an acute and a chronic form of dextran sodium sulfate (DSS)-induced colitis. It also significantly improved histopathological scores in interleukin (IL)-10-deficient mice. This was accompanied by reductions in both the basal and lipopolysaccharide-stimulated secretion of TNF-α and interferon-γ in colonic organ cultures from IL-10 −/− mice. In this model, efficacy was obtained with both a prophylactic treatment regimen or a therapeutic dosing protocol begun after colitis was already present. In both the DSS and IL-10 −/− models, scrambled and mismatch control oligonucleotides were largely without effect, suggesting that ISIS 25302 was exerting its effects through a sequence-dependent antisense mechanism.