The inclusion of high-quality hay (HQH), in place of concentrates, shifts dietary carbohydrate and the extent by which these shifts effect epimural microbiota and epithelial gene expression of the rumen has not yet been evaluated. Eight ruminally cannulated non-lactating Holstein cows were used in a replicated 4 × 4 Latin square design with four dietary treatments containing HQH, with either 0% (100% HQH), 25% (75% HQH) or 40% (60% HQH) concentrate. The fourth group (CON), considered as control, was fed with 60% normal fibre-rich hay and 40% concentrate. Data showed that measures of diversity for the rumen epimural population, specifically Shannon ( P = 0.004) and Simpson ( P = 0.003) indices decreased with increasing levels of HQH in the diet. The feeding of HQH shifted the epimural population from predominantly Firmicutes to Proteobacteria Phylogenetic analysis revealed that HQH feeding markedly shifted the abundance of Campylobacter from 7.8 up to 33.5% ( P < 0.001), with greater ingestion of protein (r = 0.63) and sugars (r = 0.65) in HQH diet being responsible for this shift. Expression of genes targeting for intracellular pH regulation, barrier function and nutrient uptake of rumen epithelium remained stable in spite of carbohydrate source. In conclusion, the data suggest strong alterations of ruminal epimural microbiota in response to changes of nutritive patterns of the diet. Further research is warranted to evaluate long-term effects of these significant microbial changes on rumen health and food safety aspects in cattle at a transcriptional level.