The molecular mechanisms underlying rumen epithelial adaption to high-grain (HG) diets are unknown. To gain insight into the metabolic mechanisms governing epithelial adaptation, mature nonlactating dairy cattle (n = 4) were transitioned from a high-forage diet (HF, 0% grain) to an HG diet (65% grain). After the cattle were fed the HG diet for 3 wk, they returned to the original HF diet, which they were fed for an additional 3 wk. Continuous ruminal pH, ruminal short chain fatty acids, and plasma β-hydroxybutyrate were measured on a weekly basis, and rumen papillae were biopsied from the ventral sac to assess alterations in mRNA expression profiles. The subacute form of ruminal acidosis was diagnosed during the first week of the HG period (4.6 ± 1.6 h/day <pH 5.6), but not during weeks 2 and 3, thereby indicating ruminal adaption to the HG diet. Changes in the mRNA expression profile of rumen papillae were initially examined using Bovine Affymetrix microarrays; a total of 521 differentially expressed genes (false discovery rate P < 0.08) were uncovered from the first to third week of the HG period. Ingenuity Pathway Analysis of microarray results revealed that enzymes involved in cholesterol synthesis were coordinately downregulated from the first to third week of the HG period. In addition, the LXR/RXR activation pathway was significant and included several genes involved in intracellular cholesterol homeostasis. The differential expression signature of eight genes representing the key regulatory points of cholesterol homeostasis was confirmed by quantitative real-time PCR. Based upon our pathway and network results we propose a model to explain cellular events during rumen epithelial adaptation to HG diets and thus provide molecular targets that may be useful in the treatment and prevention of ruminal acidosis.