The sarcomeric protein titin is one of the main molecular components that contribute to the passive stiffness of striated muscle tissue. In mammalian heart, Titin-based myofilament stiffness is an important determinant of myocardial distensibility and diastolic function. The passive mechanical properties of cardiac titin depend on the expression ratio of the isoform N2BA (~3.2 - 3.7 MDa, compliant) and N2B (~3.0 MDa, stiffer) and can be dynamically modified by phosphorylation. Importantly, phosphorylation by PKA and PKG reduces titin-based myofilament stiffness, whereas PKC increases it. We used cardiac samples from diabetic and non-diabetic patients that underwent cardiac surgery due to coronary artery disease and studied the influence of altered insulin homeostasis on cardiac titin isoform-composition and phosphorylation. In diabetic hearts expression of the compliant titin N2BA-isoform was increased in patients with HbA1c > 7.0%. Western blot analyses using phosphosite-directed antibodies further demonstrated a diabetes-related increase in the relative phosphorylation of the PEVK-region, and a decreased phosphorylation of the N2B-region of titin. Both alterations are known to increase titin-based myofilament stiffness. We further characterized insulin-dependent modification of titin using emryonic and adult rat cardiomyocytes. In embryonic rat cardiomyocytes insulin-treatment for 7d significantly enhanced the developmental-dependent isoform shift towards stiffer titin isoforms, and increased the relative expression of stiff N2B-titin by >12% compared to untreated cells. Furthermore, in embryonic and adult cardiomyocytes insulin acutely increased titin phosphorylation within 15 min. of treatment by initiating PKC-and PKG-activating pathways. We conclude that insulin is a potent modulator of cardiac titin, and may play an important role in modifying myofilament stiffness in human hearts.