Although fluoxetine, a selective serotonin reuptake inhibitor, is known to demonstrate anti-inflammatory activity, little information is available on the effect of fluoxetine regarding intestinal inflammation. This study investigates the role of fluoxetine in the attenuation of acute murine colitis by suppression of the NF-κB pathway in intestinal epithelial cells (IEC). Fluoxetine significantly inhibited activated NF-κB signals and the upregulated expression of interleukin-8 (IL-8) in COLO 205 colon epithelial cells stimulated with tumor necrosis factor-α (TNF-α). Pretreatment with fluoxetine attenuated the increased IκB kinase (IKK) and IκBα phosphorylation induced by TNF-α. In a murine model, administration of fluoxetine significantly reduced the severity of dextran sulfate sodium (DSS)-induced colitis, as assessed by the disease activity index, colon length, and histology. In addition, the DSS-induced phospho-IKK activation, myeloperoxidase activity, a parameter of neutrophil accumulation, and the secretion of macrophage-inflammatory protein-2, a mouse homolog of IL-8, were significantly decreased in fluoxetine-pretreated mice. Moreover, fluoxetine significantly attenuated the development of colon cancer in mice inoculated with azoxymethane and DSS. These results indicate that fluoxetine inhibits NF-κB activation in IEC and that it ameliorates DSS-induced acute murine colitis and colitis-associated tumorigenesis, suggesting that fluoxetine is a potential therapeutic agent for the treatment of inflammatory bowel disease.