Objective We aimed to investigate the involvement of the endocytosis of occludin, a key component of tight junction (TJ), in the ethanol-induced disassembly of TJ in a model of alcoholic steatohepatitis. Methods Wild-type mice were fed an ethanol-containing or isocaloric liquid diet for 8 weeks and then assessed for liver injury (histopathology and measurement of serum enzymes), gut permeability (in vivo lactulose/mannitol and ex vivo dye leakage assays), intestinal epithelium ultrastructure (transmission electron microscopy), and intestinal occludin localization (immunofluorescence microscopy). The human intestinal epithelial cell line Caco-2 was also analyzed in vitro for the effects of ethanol on the barrier function (transepithelial electrical resistance), occludin localization (immunofluorescence microscopy and Western blotting), and endocytosis pathways (double-labeling immunofluorescence microscopy with selective pathway inhibitors). Results The ethanol-fed mice developed steatohepatitis and displayed intestinal barrier dysfunction, the disruption of intestinal TJ, and enhanced intestinal endocytosis of occluding compared with the control mice. In the Caco-2 monolayers, ethanol treatment decreased transepithelial electrical resistance, disrupted TJ formation, and enhanced occludin endocytosis in a dose- and time-dependent manner. These deleterious events were reversed by pretreating the Caco-2 cells with a selective pharmacological inhibitor of macropinocytosis, but not with the inhibitors of clathrin or caveolin-mediated endocytic pathways. Conclusion Chronic ethanol exposure may increase intestinal permeability by inducing the micropinocytosis of occludin, resulting in the disruption of intestinal TJ.