Free fatty acids (FFA) are liberated from triglyceride-rich lipoproteins by lipoprotein lipase (LPL) and are considered to be a principal energy source for the heart. The peroxisome proliferator-activated receptor alpha (PPARα) is a key regulator of FFA catabolism. To investigate its role in cardiac muscle metabolism, transgenic mice overexpressing LPL in skeletal and cardiac muscle were bred on a PPARα knockout background. Fifty-five percent of male animals lacking PPARα and overexpressing LPL died within 4 months after birth. In contrast, females of this genotype stayed alive. Deceased animals exhibited cardiopulmonary congestion but had no increase of neutral lipids in the heart. Changes in plasma glucose, FFA, lactate, and triglycerides did not clearly account for gender-specific differences in mortality; however, they indicated a critical role for PPARα during fasting. Analysis of cardiac function revealed that in isolated perfused hearts, left ventricular developed pressure (a measure of contractility) was markedly lower in PPARα knockout mice overexpressing LPL compared with controls. Glucose uptake of isolated perfused hearts was significantly higher in PPARα knockout mice with both normal or increased LPL expression. However, uptake of FFA was not different among genotypes. In contrast, fasted FFA levels were significantly lower in cardiac muscle of PPARα knockout mice with normal LPL expression (− 26%) and PPARα knockout mice overexpressing LPL (− 38%) compared with controls. Our results indicate a critical role for PPARα in myocardial pump function and suggest that mouse models combining different genetic effects such as PPARα knockout mice overexpressing muscle LPL may be useful to study cardiomyopathies.