minal epithelium adapts to dietary change with well-coordinated alterations in metabolism, proliferation, and permeability. To further understand the molecular events controlling diet effects, the aim of this study was to evaluate protein expression patterns of ruminal epithelium in response to various feeding regimes. Sheep were fed with a concentrate-supplemented diet for up to 6 wk. The control group received hay only. Proteome analysis with differential in gel electrophoresis technology revealed that, after 2 days, 60 proteins were significantly modulated in ruminal epithelium in a comparison between hay-fed and concentrate-fed sheep (P Ͻ 0.05). Forty proteins were upregulated and 20 proteins were downregulated in response to concentrate diet. After 6 wk of this diet, only 14 proteins were differentially expressed. Among these, 11 proteins were upregulated and 3 downregulated. To identify proteins that were modulated by dietary change, two-dimensional electrophoresis was coupled with liquid chromatography electrospray ionization mass spectrometry. The differential expression of selected proteins, such as esterase D, annexin 5, peroxiredoxin 6, carbonic anhydrase I, and actin-related protein 3, was verified by immunoblotting and/or mRNA analysis. The identified proteins were mainly associated with functions related to cellular stress, metabolism, and differentiation. These results suggest new candidate proteins that may contribute to a better understanding of the signaling pathways and mechanisms that mediate rumen epithelial adaptation to high-concentrate diet.proteomics; mRNA expression; protein markers; sheep RUMINANTS ARE OF MAJOR WORLDWIDE interest and economic importance because of increasing meat and milk production. A substantial improvement in the productivity and performance of ruminants can only be achieved with adequate dietary supplementation. When high-yielding cattle, milk sheep, or goats merely consume forage, the intake of energy and protein is too low for the desired rates of animal performance. Hence, supplementation of forage-fed ruminants with sufficient amounts of energy and protein is necessary and significantly improves milk production or daily weight gain (41), but, particularly, a surplus of easily fermented carbohydrates or low-fiber intake will place the animals at risk. Such a change in the diet is a challenge for rumen microbes (31) and especially for the ruminal epithelium with respect to adaptation to the new fermentation pattern (47,55). Insufficient adaptation to diet may result in disintegration of the epithelium (22), translocation of lipopolysaccharides (30), and disturbed transport mechanisms (22) and can further cause subacute ruminal acidosis (14, 49). This disease not only can lead to depressed feed intake and milk production, but can also be associated with laminitis, inflammation, and liver abscesses (14,42,49).Hence, one objective of attaining an adequate diet composition for ruminants is to maintain the physiological functions of the forestomach, such as optimal fe...