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The hindgut epithelial barrier plays an important role in maintaining absorption and immune homeostasis in ruminants. However, little information is available on changes in colon epithelial barrier structure and function following grain-induced subacute ruminal acidosis (SARA). The objective of this study was to investigate the effects of grain-induced SARA on colon epithelial morphological structure, permeability, and gene expression involved in epithelial barrier function. Twelve mid-lactating (136 ± 2 d in milk; milk yield = 1.68 ± 0.15 kg/d) Saanen dairy goats with 62.13 ± 4.76 kg of body weight were randomly divided into either the control (CON) treatment (n = 6) or SARA treatment (n = 6). The CON goats were fed a basal diet with a nonfiber carbohydrates to neutral detergent fiber ratio of 1.15 for 60 d. The SARA goats were fed 4 diets with increasing nonfiber carbohydrates to neutral detergent fiber ratio at 1.15, 1.49, 2.12, and 2.66 to induce SARA, with each diet (referred to as period) being fed for 15 d, including 12 d for adaptation and 3 d for sampling. Continuous ruminal pH recordings were used to diagnose the severity of SARA. Additionally, colonic tissues were collected to evaluate the epithelial morphological structure, permeability, and expression of tight junction proteins using transmission electron microscopy, Ussing chamber, quantitative real-time PCR, and Western blotting. Profound disruption in the colonic epithelium was mainly manifested as the electron density of tight junctions decreased, intercellular space widened, and mitochondria swelled in SARA goats. Colon epithelial short-circuit current, tissue conductance, and the mucosal-to-serosal flux of fluorescein isothiocyanate-dextran 4 kDa were increased and potential difference was decreased in SARA goats compared with CON goats. Subacute ruminal acidosis increased mRNA and protein expression levels of CLDN1 and OCLN in the colonic epithelium. Overall, the data of the present study demonstrate that SARA can impair the barrier function of the colonic epithelium at both structural and functional levels, which is associated with severe epithelial structural damage and increased permeability and changes in the expression of tight junction proteins.