Increased epithelial permeability is observed in inflammatory states. However, the mechanism by which inflammatory mediators such as IFN‐γ increase epithelial permeability is unknown. We recently observed that IFN‐γ induces disassembly of tight junctions (TJ); in this study we asked whether such TJ disassembly is mediated by endocytosis of junctional proteins. The role of three major internalization pathways in disruption of TJ in IFN‐γ‐treated intestinal epithelial cells was analyzed using selective inhibitors and markers of the pathways. No role for the clathrin‐ and caveolar‐mediated endocytosis in the IFN‐γ‐induced internalization of TJ proteins was observed. However, inhibitors of macropinocytosis blocked internalization of TJ proteins and junctional proteins colocalized with macropinocytosis markers, dextran and phosphatidylinositol‐3,4,5‐trisphosphate. Internalized TJ proteins were identified in early and recycling endosomes but not in late endosomes/lysosomes. These results for the first time suggest that IFN‐γ produces a leaky epithelial barrier by inducing macropinoytosis of TJ proteins. Bruewer, M., Utechm, M., Ivanov, A. I., Hopkins, A. M., Parkos, C. A., Nusrat, A. Interferon‐γ induces internalization of epithelial tight junction proteins via a macropinocytosis‐like process. FASEB J. 19, 923–933 (2005)