Alterations in rumen epithelial tight junctions (TJs) at the tissue and molecular levels during high-grain (HG) diet feeding are unknown. Here, 10 male goats were randomly assigned to either a hay diet (0% grain; n ϭ 5) or HG diet group (65% grain; n ϭ 5) to characterize the changes in ruminal epithelial structure and TJ protein expression and localization using scanning and transmission electron microscopy, quantitative real-time PCR, Western blot analysis, and immunofluorescence. After 7 wk of feeding, ruminal free LPS in HG group increased significantly (P Ͻ 0.001) compared with the hay group, and free LPS in the peripheral blood was detectable with concentrations of 0.8 Ϯ 0.20 EU/ml, while not detectable in the control, suggesting a leakage of LPS into the blood in the HG group. Correspondingly, the HG-fed goats showed profound alterations in ruminal epithelial structure and TJ proteins, depicted by marked epithelial cellular damage and intercellular junction erosion, down-regulation of TJ proteins claudin-4, occludin, and zonula occludens-1 mRNA and protein expression, as well as redistribution of claudin-1, claudin-4, and occludin. Furthermore, these changes in TJ proteins in the HG group were coupled with the upregulation of mRNA levels for the cytokines TNF-␣ and IFN-␥ in the ruminal epithelia. These results demonstrated for the first time that the HG diet feeding caused disruption of ruminal epithelial TJs that was associated with a local inflammatory response in the rumen epithelium. These findings may provide new insights into understanding the role of TJ proteins in the ruminal epithelial immune homeostasis of ruminants. rumen epithelium; high-grain diet; tight junction protein; lipopolysaccharide; inflammation IN CURRENT INTENSIVE RUMINANT production system, it has become common to use high-concentrate diets (high-grain, HG) to maximize energy intake and to improve milk production or daily weight gain, but highly fermentable diets can put the animal at risk. Rapidly fermentable nonstructural carbohydrates increase the rate of fermentation acid production in the rumen, and the accumulation of the acids leads to a decrease in ruminal pH, hyperosmolarity, and an increase in ruminal toxin concentration (2, 29). Previous studies have investigated the specific effect of low pH (9, 26), hyperosmolarity (20, 34), or an exposure to toxins (7) on ruminal epithelial barrier function in vitro. It should be acknowledged that any one or the combination of these factors may affect epithelial barrier function. In addition, the prominent histological alterations during HG feeding strongly suggest an impaired barrier function (35-37) that may provide the opportunity for the translocation of toxins and bacteria from the rumen into the blood, ultimately affecting animal health and productivity (28). Although there is fundamental knowledge about the consequences of HG feeding on ruminal epithelial barrier function, remarkably little information is currently available about the underlying molecular changes in ruminal epi...