Aberrant cholangiocyte reactions in response to inflammatory stimuli are important pathogenic factors for the persistent biliary inflammation in patients with cholangiopathies. Overexpression of intercellular cell adhesion molecule-1 (ICAM-1) in cholangiocytes is a common pathological feature in inflammatory cholangiopathies and can promote cholangiocyte interactions with effector lymphocytes in the portal region. In this study, we tested the involvement of miRNA-mediated posttranscriptional regulation in IFN-gamma-induced ICAM-1 expression in cholangiocytes. Using both immortalized and nonimmortalized human cholangiocyte cell lines, we found that IFN-gamma activated ICAM-1 transcription and increased ICAM-1 protein expression. Inhibition of ICAM-1 transcription could only partially block IFN-gamma-induced ICAM-1 expression at the protein level. In silico target prediction analysis revealed complementarity of miR-221 to the 3'-untranslated region of ICAM-1 mRNA. Targeting of ICAM-1 3'-untranslated region by miR-221 resulted in translational repression in cholangiocytes but not ICAM-1 mRNA degradation. Functional inhibition of miR-221 with anti-miR-221 induced ICAM-1 protein expression. Moreover, IFN-gamma stimulation decreased miR-221 expression in cholangiocytes in a signal transducer and activator of transcription 1-dependent manner. Transfection of miR-221 precursor abolished IFN-gamma-stimulated ICAM-1 protein expression. In addition, miR-221-mediated expression of ICAM-1 on cholangiocytes showed a significant influence on the adherence of cocultured T cells. These findings indicate that both transcriptional and miRNA-mediated posttranscriptional mechanisms are involved in IFN-gamma-induced ICAM-1 expression in human cholangiocytes, suggesting an important role for miRNAs in the regulation of cholangiocyte inflammatory responses.