Cardiac intracellular lipid accumulation (steatosis) is a pathophysiological phenomenon observed in starvation and diabetes mellitus. Perilipin 2 (PLIN2) is a lipid droplet (LD)-associated protein expressed in non-adipose tissues, including the heart. To explore the pathophysiological function of myocardial PLIN2, we generated transgenic (Tg) mice by cardiac-specific overexpression of PLIN2. Tg hearts showed accumulation of numerous small LDs associated with mitochondrial chains, and high cardiac triacylglycerol (TAG) content (8-fold greater than wild-type (Wt) mice). Despite massive steatosis, cardiac uptake of glucose, fatty acids and VLDL, systolic function, and expression of metabolic genes were comparable in the two genotypes, and no morphological changes were observed by electron microscopy in the Tg hearts. Twenty-four hours fasting markedly reduced steatosis in Tg hearts, while Wt mice showed accumulation of LDs. Although activity of adipose triglyceride lipase in heart homogenate was comparable between Wt and Tg mice, activity of hormone-sensitive lipase (HSL) was 40-50% less in Tg than Wt mice, under both feeding and fasting conditions, suggesting interference of PLIN2 with HSL. Mice generated through crossing of PLIN2-Tg mice and HSL-Tg mice showed cardiac-specific HSL overexpression and complete lack of steatosis. The results suggest that cardiac PLIN2 plays an important pathophysiological role in the development of dynamic steatosis, and that the latter was prevented by upregulation of intracellular lipases, including HSL.