The adult mammalian heart has the largest metabolic demand per gram of any organ in the body and derives 50 ? 70% of its energy from fatty acid (FA) oxidation. Within cardiomyocytes, FA are stored as triacylglycerol (TAG) and can be mobilized on demand by lipases. Adipose triglyceride lipase (ATGL) initiates TAG hydrolysis and its loss results in cardiac TAG accumulation and cardiac dysfunction. Cardiac ATGL is regulated by auxiliary proteins such as perilipin 5 and comparative gene identification 58 (CGI-58), which restrict or promote ATGL action. Recently, another regulatory protein, termed G0/G1 switch protein 2 (G0S2), was shown to inhibit ATGL in liver and adipose tissue. G0S2 is also expressed in the heart indicating a role in the regulation of cardiac TAG turnover. To investigate whether G0S2 modulates cardiac TAG metabolism in vivo, we generated transgenic mice overexpressing or lacking G0S2, respectively. Global loss of G0S2 did not change cardiac TAG content indicating that G0S2-deficiency does not affect cardiac TAG turnover. In contrast, cardiac-specific overexpression of G0S2 (G0S2Tg) resulted in pronounced cardiac steatosis resembling the phenotype of ATGL-deficient mice (ATGL-/-). G0S2 was found to physically interact with ATGL in G0S2Tg heart lysates and resulted in decreased TAG hydrolase activity in vitro suggesting impaired ATGL activity. Strikingly, cardiac steatosis was accompanied by increased expression of pro-inflammatory cytokines and severe interstitial fibrosis in ATGL-/- mice, but not in G0S2Tg. Consequently, G0S2Tg did not develop cardiac dysfunction such as occurs in ATGL-/- mice. Collectively, these results demonstrate that overexpression of G0S2 in the heart inhibits ATGL activity and results in pronounced cardiac steatosis whereas global loss of G0S2 does not affect cardiac TAG homeostasis.