Bovine skeletal muscle is a tissue of significant value to the beef industry and global economy. Proteomic analyses offer the opportunity to detect molecular mechanisms regulating muscle growth and intramuscular fat accumulation. The current study aimed to investigate differences in protein abundance in skeletal muscle tissue of cattle from two breeds of contrasting maturity (early vs. late maturing), adiposity, and muscle growth potential, namely, Belgian Blue (BB) x Holstein Friesian and Aberdeen Angus (AA) x Holstein Friesian. Twenty AA (n = 10) and BB (n = 10) sired steers, the progeny of sires of either high or low genetic merit, expressed as expected progeny difference for carcass weight (EPDcwt), and bred through AI, were evaluated as 4 genetic groups, BB-High, BB-Low, AA-High, and AA-Low (n = 5 per treatment). Chemical composition analysis of M. longissimus lumborum showed greater protein and moisture and decreased lipid concentrations for BB-sired compared with AA-sired steers. To investigate the effects of both sire breed and EPDcwt on M. longissimus lumborum, proteomic analysis was performed using 2-dimensional difference gel electrophoresis followed by mass spectrometry. Proteins were identified from their peptide sequences, using the National Center for Biotechnology Information (NCBI) and Swiss-prot databases. Metabolic enzymes involved in glycolysis (glycogen phosphorylase, phosphoglycerate mutase) and the citric acid cycle (aconitase 2, oxoglutarate dehydrogenase) were increased in AA- vs. BB-sired steers. Expression of proteins involved in cell structure, such as myosin light chain isoforms and troponins I and T, were also altered due to sire breed. Furthermore, heat shock protein beta-1 and peroxiredoxin 6, involved in cell defense, had increased abundance in muscle of AA-sired relative to BB-sired steers. Protein abundance of glucose-6-phosphate isomerase, enolase-3, and pyruvate kinase was greater in AA-sired animals of High compared with Low EPDcwt. Changes in the expression of these proteins were supported by gene expression analysis using quantitative real-time PCR. This information will aid in our understanding of genetic influences controlling muscle growth and fat accumulation and could contribute to future breeding programs to increase lean tissue gain of beef cattle.