Oxidative stress is defined as an imbalance between the antioxidant defense system and the production of reactive oxygen species (ROS). At low levels, ROS are involved in the regulation of redox signaling for cell protection. However, upon chronical increase of oxidative stress, cell damage occurs due to protein, DNA and lipid oxidation. Here we investigated oxidative modifications of myofilament proteins and its role in modulating cardiomyocyte function in end stage human failing hearts. We found altered maximum Ca2+-activated tension and Ca2+-sensitivity of force production of skinned single cardiomyocytes in end-stage human failing hearts compared to non-failing hearts, which was corrected upon treatment with reduced glutathione enzyme. This was accompanied by increased oxidation of troponin I and myosin binding protein C and de-creased levels of protein kinases A (PKA) and C (PKC)-mediated phosphorylation of both proteins. The Ca2+-sensitivity and maximal tension correlated strongly with myofilament oxidation levels, hypo-phosphorylation, and oxidative stress parameters measured in all samples. Furthermore, we detected elevated titin-based myocardial stiffness in HF myocytes which was reversed by PKA and reduced glutathione enzyme treatment. Finally, many oxidative stress and inflammation parameters were significantly elevated in failing compared to non-failing hearts and corrected upon treatment with the antioxidant GSH enzyme. Here we provide evidence that altered mechanical properties of failing human cardiomyocytes is partially due to phosphorylation, S-glutathionylation, and the interplay between the two post-translational modifications, which contribute to development of heart failure.