ATP and creatine phosphate (PCr) are prime myocardial high-energy phosphates. Their relative concentrations are conserved among mammalian species and across a range of physiologic cardiac workloads. The cardiac PCr/ATP ratio is decreased with several pathologic conditions, such as ischemia and heart failure, but there are no reports of an increase in the cardiac PCr/ATP ratio in any species or with interventions. We studied the in vivo energetics in transgenic mice lacking expression of the glucose transport protein GLUT4 (G4N) and observed a significant 60% increase in the myocardial PCr/ATP ratio in G4N that was confirmed in three different experimental settings including intact animals. The higher PCr/ATP in G4N is cardiac-specific and is due to higher total cardiac creatine (CR) concentrations in G4N than in wild-type (WT). However, [ATP], [ADP], and -DG(-ATP) did not differ between the strains. Expression of the creatine transport protein (CreaT) that is responsible for creatine uptake in myocytes was preserved in G4N cardiac tissue. These observations demonstrate, for the first time to our knowledge, that G4N manifest a unique increase in the cardiac PCr/ATP ratio, which suggests a novel genetic strategy for increasing myocardial creatine levels.