The sarcomeric protein titin is one of the main molecular components that contribute to the passive stiffness of muscle tissue. The passive mechanical properties of cardiac titin depend in part on the expression ratio of the isoforms N2BA (~3,2 - 3,7 MDa, compliant) and N2B (~3,0 MDa, stiffer), and can be dynamically modified by phosphorylation through PKA, PKG, CaMK and PKC. Importantly, phosphorylation by PKA and PKG reduces titin-based myofilament stiffness, whereas PKC increases it. To elucidate how titin domain phosphorylation is controlled we studied phosphorylation of the titin-PEVK region at Ser11878 (UniProtKB Acc. #Q8WZ42) using embryonic rat cardiomyocytes. Titin isoform composition and phosphorylation was analyzed by Coomassie-stained 1.9% SDS PAGE and Western blot analyses using a phosphosite-specific titin-PEVK antibody. Interestingly, insulin application significantly increased titin PEVK phosphorylation by up to 5,25 fold within 45 min. of treatment. This effect was blocked by inhibition of the PI3K-pathway (LY294002), suggesting insulin-mediated phosphorylation of titin by PDK1/PKC_alpha. Furthermore, we studied the effects of metformin, an oral antidiabetic drug for the treatment of type 2 diabetes, on titin-PEVK Ser11878. We found that metformin treatment also increased titin-PEVK phosphorylation and activated the PI3K depending PDK1/PKC_alpha pathway. We conclude that insulin as well as the therapeutic drug metformin strongly affect cardiac titin, and may therefore play an important role in modifying myocardial stiffness in human hearts.