The important role of intestinal tryptophan metabolism in regulating gut homeostasis though activation of aryl hydrocarbon receptor (AhR) has been increasingly reported. In order to explore the relationship between intestinal tryptophan metabolism, intestinal AhR activation and onset of inflammatory bowel disease (IBD), fecal samples were collected from 20 healthy controls (HC) and 80 IBD patients: ulcerative colitis (UC) in active status (n=20) and remission (n=20), Crohn’s disease (CD) in active status (n=20) and remission (n=20). Human colonic epithelial cells (Caco-2) were exposed to fecal water (FW, aqueous phase of feces) to measure AhR activation level. Ten tryptophan metabolites which were reported to be AhR ligands were quantified in FW by liquid chromatography-mass spectrometry. The FW derived from IBD patients induced significantly lower AhR activation in Caco-2 cells, when compared to FW from HC. Moreover, AhR activation ability of FW was negatively associated with intestinal inflammation, as indicated by the fecal calprotectin level. The tryptophan metabolite indole was significantly increased in active CD patients, while indole-3-acetic acid (IAA) and serotonin were significantly decreased in IBD patients, when compared to HC. In addition, FW with higher AhR activation ability, contained increased concentrations of tryptophan and kynurenine, whereas IAA and serotonin levels were reduced. We further compared the AhR activation ability of original FW and a mixture of 10 tryptophan metabolites with relevant concentrations measured in the FW, to see how much AhR activation of FW can be explained by these ligands. Unexpectedly, these ten ligands did not induced obvious AhR activation, when compared to original FW. These results suggest that aberrant tryptophan metabolism and intestinal AhR activation were involved in IBD pathogenesis. Tryptophan metabolites are correlated to intestinal AhR activation, but they can not explain the AhR activation ability of FW. The dominant fecal AhR agonists still need be further identified.