Aims: Isoform-switching of the elastic protein titin is a main mechanism for adjusting passive myocardial stiffness in perinatal heart development and chronic heart disease. Evidence suggested that thyroid hormone signaling converging onto the phosphoinositol-3-kinase (PI3K)/AKT pathway is an important determinant of the cardiac titin-isoform pattern in developing cardiomyocytes. We hypothesized that other activators of PI3K/AKT, particularly insulin, may similarly alter the titin-isoform composition, thereby modifying titin-based stiffness. Methods: Embryonic rat cardiomyocytes were cultured in the presence of insulin for seven days and titin-isoform expression was analyzed by 2% SDS-PAGE. We further tested the effect of insulin-deficiency on titin-isoform expression and titin-based passive stiffness in streptozotocin-treated (STZ) rats as a model for diabetes mellitus (type 1). Results: After seven days in culture the mean proportion of the stiff N2B titin isoform (3,000 kDa) was significantly increased from 53% in control cells to 64% of total titin in insulin-treated cells. This insulin-dependent titin-isoform shift was blocked in the presence of PI3K-inhibitor, LY294002, suggesting that insulin regulates the cardiac titin-isoform pattern by activating the PI3K/AKT pathway. Within four months, STZ rats developed cardiac hypertrophy and mild left ventricular (LV) fibrosis, concomitant with elevated glucose levels. The mean proportion of N2B-titin was significantly decreased from 86% in control LV to 78% in LV of STZ rats. Wormlike chain modeling of titin elasticity suggested that such a change reduces titin-based passive stiffness by ~6%. Results of mechanical measurements on skinned cardiac fiber bundles confirmed minor passive stiffness modifications in STZ myocardium. Conclusion: We conclude that insulin signaling regulates titin-isoform composition in cardiac development and could also contribute to altered diastolic function in diabetic cardiomyopathy.