While the balance between carbohydrates and fatty acids for energy production appears to be crucial for cardiac homeostasis, much remains to be learned about the molecular mechanisms underlying this relationship. Given the reported benefits of cGMP signaling on the myocardium, we investigated the impact of its chronic activation on cardiac energy metabolism using mice overexpressing a constitutively active cytoplasmic guanylate cyclase (GC+/0) in cardiomyocytes. Ex vivo working GC+/0 heart perfusions with 13C-labeled substrates revealed an altered pattern of exogenous substrate fuel selection compared to controls, namely a 38 ± 9% lower contribution of exogenous fatty acids to acetyl-CoA formation, while that of carbohydrates remains unchanged despite a two-fold increase in glycolysis. The lower contribution of exogenous fatty acids to energy production is not associated with changes in energy demand or supply (contractile function, oxygen consumption, tissue acetyl-CoA or CoA levels, citric acid cycle flux rate) or in the regulation of β-oxidation (acetyl-CoA carboxylase activity, tissue malonyl-CoA levels). However, GC+/0 hearts show a two-fold increase in the incorporation of exogenous oleate into triglycerides. Furthermore, the following molecular data are consistent with a concomitant increase in triglyceride hydrolysis: (i) increased abundance of hormone sensitive lipase (HSL) protein (24 ± 11%) and mRNA (22 ± 4%) as well as (ii) several phosphorylation events related to HSL inhibitory (AMPK) and activation (ERK 1/2) sites, which should contribute to enhance its activity. These changes in exogenous fatty acid trafficking in GC+/0 hearts appear to be functionally relevant, as demonstrated by their resistance to fasting-induced triglyceride accumulation. While the documented metabolic profile of GC+/0 mouse hearts is partly reminiscent of hypertrophied hearts, the observed changes in lipid trafficking have not been previously documented, and may be part of the molecular mechanism underlying the benefits of cGMP signaling on the myocardium. © 2008 Elsevier Inc. All rights reserved.