Bacteroides fragilis produces an approximately 20‐kDa heat‐labile toxin (B. fragilis enterotoxin, BFT) which is known to be associated with diarrhea. To determine whether cyclooxygenase (COX)‐2, via NF‐κB activation, can contribute to BFT‐induced diarrhea, the relationship between COX‐2 expression and fluid secretion in BFT‐stimulated human intestinal epithelial cells was examined. BFT stimulation increased the expression of COX‐2, but not COX‐1, in human intestinal epithelial cells. Suppression of the NF‐κB signal significantly decreased COX‐2 expression in response to BFT stimulation. Prostaglandin E₂ (PGE₂) levels were increased in parallel with COX‐2 expression, and, conversely, PGE₂ production was significantly inhibited when COX‐2 or NF‐κB activities were suppressed using COX‐2 small interfering RNA (siRNA), p65 NF‐κB subunit siRNA, or a retrovirus encoding the IκBα superrepressor. In addition, a selective COX‐2 inhibitor, NS‐398, significantly inhibited the increased cAMP level induced by BFT stimulation. Furthermore, a selective COX‐2 inhibitor prevented BFT‐induced PGE₂ production and ileal fluid secretion in a mouse ileal loop model. These results suggest that the secretory response to BFT stimulation may be mediated by the production of PGE₂, through NF‐κB activation and the up‐regulation of COX‐2 in intestinal epithelial cells.