In obese rodents, excess myocardial lipid accumulation (lipotoxicity) of myocardium may cause cardiomyopathy that in the obese Zucker diabetic fatty (ZDF) fa/fa rat can be prevented by treatment with troglitazone (TGZ). To determine the underlying mechanisms, we measured total 5′-AMP-activated kinase (AMPK) protein and its activated, phosphorylated form, P-AMPK. P-AMPK was significantly reduced in both ZDF fa/fa rat and ob/ob mouse hearts compared with lean, wild-type controls. TGZ treatment of obese ZDF rats, which lowered cardiac lipid content, increased P-AMPK. Expression of protein phosphatase 2C (PP2C), which inactivates AMPK activity by dephosphorylation, was increased in untreated ZDF fa/fa rat hearts, but fell with TGZ treatment, suggesting that PP2C can influence AMPK activity. In cultured myocardiocytes, fatty acids reduced P-AMPK, suggesting a feed-forward effect of lipid overload. Our findings highlight a role of PP2C and AMPK in the derangements of cardiac lipid metabolism in obesity and provide new insights as to the mechanisms of the liporegulatory disorder leading to lipotoxic cardiomyopathy.