Rumen is important to digest forage constituting a critical step to supply enough nutrients for animalsgrowth and production.In this study, we hypothesize that rumen may function differently in grass-and grain-fed regimens.The objective of this study was to find the differentially expressed genes in the ruminal wall of grass-fed and grain-fed steers, and then explored the potential biopathways.Thereby, the RNA Sequencing(RNA-Seq) method was used to measure the gene expression level in the ruminal wall.The analysis totally detected 342 differentially expressed genes between ruminal wall samples of beef raised under different regimens.The Ingenuity Pathway Analysis (IPA) analysis found 13 significantly enriched pathways, most of which were related to cell development and biosynthesis.Our results provided valuable insights into the molecular mechanisms resulting in the phenotype difference between grass-fed and grain-fed cattle.