Heart failure is a leading cause of morbidity and mortality in the USA and is closely associated with diabetes mellitus. The molecular link between diabetes and heart failure is incompletely understood. We recently demonstrated that insulin receptor substrate 1, 2 (IRS1, 2) are key components of insulin signaling and their dysfunction mediates insulin resistance, resulting in metabolic dysregulation and heart failure. Loss of IRS1 and IRS2 is associated with downstream Akt inactivation and in turn activation of the forkhead transcription factor Foxo1. To determine the role of Foxo1 in control of heart failure in insulin resistance and diabetes, we generated mice lacking Foxo1 gene specifically in the heart. Mice lacking both IRS1 and IRS2 in adult hearts exhibited severe heart failure, loss of mitochondria, and a remarkable increase in the β-isoform of myosin heavy chain (β-MHC) gene expression, while deletion of cardiac Foxo1 gene largely prevented the heart failure and the loss of mitochondria, and resulted in a decrease in β-MHC expression. The effect of Foxo1 deficiency on rescuing cardiac dysfunction was also observed in db/db mice and high-fat diet (HFD) mice. Using cultures of primary ventricular cardiomyocytes, we found that Foxo1 interacts with the promoter region of β-MHC and stimulates gene expression, mediating an effect of insulin that suppresses β-MHC expression. Taken together, our study suggests that Foxo1 has important roles in promoting diabetic cardiomyopathy and controls β-MHC expression in development of cardiac dysfunction. Targeting Foxo1 and its regulation will provide novel strategies in preventing metabolic and myocardial dysfunction and influencing MHC plasticity in diabetes mellitus.