Intramyocellular triacylglycerol (IMTG) accumulation is strongly associated with insulin resistance and metabolic complications of obesity, whereas comparable IMTG accumulation in endurance-trained athletes is associated with insulin sensitivity (the athlete's paradox). The role of IMTG hydrolysis in these processes remains unclear. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating intracellular TG hydrolysis, and is therefore a critical determinant of storage/production of toxic as well as essential lipid metabolites. ATGL is highly expressed and functional in skeletal muscle - quantitatively the most important site for insulin-stimulated glucose disposal and fatty acid oxidation, and yet the physiological relevance of ATGL action within skeletal muscle remains unknown. To determine the contribution of ATGL-mediated TG hydrolysis within skeletal muscle to tissue-specific and systemic glucose homeostasis and insulin action, we generated animal models with decreased (Skeletal Muscle-specific ATGL KnockOut or SMAKO mice) and increased (Ckm-Atgl transgenic mice) ATGL action exclusively in skeletal muscle resulting in increased and decreased IMTG, respectively. Interestingly, despite dramatic changes in IMTG content, modulation of ATGL action in skeletal muscle did not significantly influence systemic energy, lipid, or glucose homeostasis. Furthermore, changes in intracellular lipids were not associated with muscle-specific changes in insulin action or mitochondrial function. Similar results were obtained in mice fed chow or high-fat diet. Thus, modulation of ATGL action in skeletal muscle alters IMTG content but does not influence metabolic phenotypes in response to nutritional challenges, suggesting that ATGL action may be more critical during functional stress (i.e. exercise) rather than metabolic/nutritional stress.