ABSTRACT: An abrupt transition from high forage to high fermentable diet may induce digestive disorders in the rumen. To date, the host mechanisms regulate the adaption to such dietary transition are largely unknown. To understand the molecular mechanisms involved in such phenomena, RNA-sequencing was performed to identify the changes in the transcriptomes of ruminal epithelia during a rapid high grain transition from 3% to 92% grain. In total, the expression of 11,044, 11,322 and 11,282 genes was detected in ruminal epithelia of beef heifers (n=15) fed 3%, 75% and 92% barley grain diet, respectively. When diet was changed from 75% to 92% grain, a significant difference was observed in the mean ruminal pH change among the individuals with five heifers decreased (DG; pH from 6.30 ± 0.09 to 5.87 ± 0.15, P < 0.01) and five other ones increased (UG; pH from 5.84 ± 0.42 to 6.35 ± 0.37, P < 0.05). In the ruminal epithelia of DG animals, the expression of genes involved in ketogenesis (HMGCL, P < 0.1) and lipid synthesis (SREBF2, P < 0.1; FABP4, P < 0.05) was increased, the abundance of genes involved in short chain fatty acid transporters (SMCT1, P < 0.1) and monocarboxylate transporters (MCT4, P < 0.1) was decreased. The function of “Concentration of lipid” was activated after 75% to 92% grain diet transition. Comparing to DG animals, the expression of ketogenesis (ACAT2, P < 0.05; HMGCS P < 0.1) and cholesterol synthesis related genes (HMGC and FDPs, P < 0.1) was deceased, while the expression of proton and cholesterol efflux related genes NHE3 and ABCA1 were increased (P < 0.05), and the function of “Storage of lipid” was inhibited in the ruminal epithelial of UG heifers. In addition, the function of differentially expressed (DE) genes in DG were involved in “T cell receptor signaling” and “complement and coagulation cascades” pathways, while the functions “metabolism of xenobiotics by cytochrome P450” and “p53 signaling pathway” were enriched for DE genes in UG animals. Furthermore, the single nucleotide polymorphisms (SNP) discovery by RNA-seq was performed, and SNP (g.46834311A > G) in FABP4 identified between two groups of animals (P < 0.1). The expression of genes involved in regulating lipid transport and fatty acid metabolism varied between DG and UG heifers during rapid 75% to 92% grain transition. The identified genes and SNP could be potential markers and may account for the varied ruminal pH responses between DG and UG heifers, which can be potentially applied in breeding and selecting beef cattle with more tolerance in dietary transition stress.