Tenderness constitutes one of the main characteristics in beef quality.In this study, a methylome analysis was performed in closed Wye Angus steers with genetically homogenous phenotypic backgrounds, to further understand the regulation of gene expression and the relationship between DNA methylation and beef tenderness.Through the use of the Methyl CpG Binding Domain protein-enriched genome sequencing (MBD) method, differentially methylated regions (DMRs) were identified and annotated, respectively.Among 1,576 identified DMRs, 983 were downregulated and 593 were upregulated in the "tender" group, demonstrating a global decrease in DNA methylation.Finally, we found some epigenetically-regulated genes and important pathways related to beef tenderness, including calcium signaling, heparin sulfate biosynthesis, Gamma-Amino Butyric Acid (GABA) receptor signaling, mitochondrial L-carnitine shuttle and lipopolysaccharide/interleukin 1 (LPS/IL-1).The results from the research provide new insights to the regulation of genes that affect beef tenderness though DNA methylation.