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A substantial improvement in animal performance has been primarily achieved by feeding ruminants a concentrate-supplemented diet. Diet-dependent alterations in the rumen epithelium have been widely investigated, but the underlining molecular mechanisms are not yet well understood. The aim of this study has been to evaluate protein expression patterns of rumen epithelium in response to various feeding regimes. Sheep were fed with a concentrate-supplemented diet for up to six weeks. The control group received hay only. Proteome analysis with differential in gel electrophoresis (DIGE) technology revealed that, after 2 days, 60 proteins were significantly modulated in rumen epithelium in a comparison between hay-fed and concentrate-fed sheep. After 6 weeks of this diet, 14 proteins were differentially expressed suggesting a time-dependent adaptation of the rumen epithelium. In order to identify proteins that were modulated by dietary change, two-dimensional electrophoresis was coupled with liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS). The identified proteins were mainly associated with functions related to cellular stress, metabolism, and differentiation. 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. These findings open new perspectives for further studies of diet-dependent adaptation in sheep rumen epithelium.