In order to study the molecular mechanism involved in meat quality differences especially tenderness variance caused by castration in Qinchuan cattle males, this study investigated the gene expression profile of Longissimus thoracis (LT) muscle and screened differentially expressed genes in LT muscle from both intact male and castrated male Qinchuan cattle at 36months of age utilising Bovine Genome Array. Significance Analysis of Microarrays (SAM) was used to identify the differentially expressed genes, Go (Gene Ontology) and pathway analyses were conducted on which by a free Web-based Molecular Annotation System 2.0 (MAS 2.0). Approximately 11,000 probe sets representing 10,000 genes were detected in LT muscle of 36-month-old Qinchuan cattle. After SAM analysis of the microarray data, 142 genes were shown to be differentially expressed. These genes were predominantly involved in collagen fibril organization and synthesis, regulation of cell growth and development and striated muscle contraction. The significant pathways involved mainly included ECM–receptor (extracellular matrix-receptor) interaction, cell communication and focal adhesion. Quantitative real-time PCR (qRT-PCR) was performed to validate some differentially expressed genes identified by microarray. These patterns of gene expression may contribute to understanding the molecular mechanism of better meat quality of beef derived from castrates than from intact males.