In this paper, we report the full-length coding sequence of bovine ATGL cDNA and analyze its expression in bovine tissues. Similar to human, mouse, and pig ATGL sequences, bovine ATGL has a highly conserved patatin domain that is necessary for lipolytic function in mice and humans. This suggests that ATGL is functionally intact as a triglyceride lipase in cattle. Tissue distribution of ATGL gene expression was highest in fat and muscle (skeletal and cardiac) tissue, while protein expression was solely detectible in the adipose tissue. The effect of 109 days of flaxseed supplementation on ATGL and adipocyte fatty acid-binding protein (FABP4 or A-FABP, E-FABP or FABP5) expression was examined in Angus steers. Supplemented steers had greater triacylglycerol (TAG) content in the muscle compared with unsupplemented ones. Additionally, supplementation increased A-FABP expression and decreased stearoyl-CoA desaturase 1 (SCD-1) expression in muscle, while total ATGL expression was unaffected. In summary, supplementation of cattle rations with flaxseed increased muscle TAG concentrations attributed in part to increased expression of key enzymes involved in lipid trafficking (A-FABP) and metabolism (SCD-1).