A role for cytokine regulated proteins in epithelial cells has been suggested in the pathogenesis of inflammatory bowel diseases (IBD). The aim of this study was to identify such cytokine regulated targets using a proteomic functional approach. Protein patterns from ³⁵S‐radiolabeled homogenates of cultured colon epithelial cells were compared before and after exposure to interferon‐γ, interleukin‐1β and interleukin‐6. Proteins were separated by two‐dimensional polyacrylamide gel electrophoresis. Both autoradiographies and silver stained gels were analyzed. Proteins showing differential expression were identified by tryptic in‐gel digestion and mass spectrometry. Metabolism related proteins were also investigated by Western blot analysis. Tryptophanyl‐tRNA synthetase, indoleamine‐2,3‐dioxygenase, heterogeneous nuclear ribonucleoprotein JKTBP, interferon‐induced 35kDa protein, proteasome subunit LMP2 and arginosuccinate synthetase were identified as cytokine modulated proteins in vitro. Using purified epithelial cells from patients, overexpression of indoleamine‐2,3‐dioxygenase, an enzyme involved in tryptophan metabolism, was confirmed in Crohn’s disease as well as in ulcerative colitis, as compared to normal mucosa. No such difference was found in diverticulitis. Potentially, this observation opens new avenues in the treatment of IBD.