Titin-based passive stiffness is an important determinant of myocardial diastolic function, and is modulated by i) alterations in the relative expression levels of the cardiac titin isoforms, N2B (3.0 MDa) and N2BA (> 3.2 MDa), and ii) phosphorylation of elastic I-band domains of titin. Importantly, phosphorylation of the N2-B unique sequence (N2-Bus) by cGMP-dependent protein kinase (PKG) or cAMP-dependent protein kinase (PKA) decreases titin stiffness, whereas phosphorylation of the titin PEVK domain by PKC increases it. Here, we analyzed whether the phosphorylation status of the titin domains is altered in human failing hearts and affects passive myofilament stiffness .Using mass spectrometry we identified novel PKG- and PKA-dependent phosphorylation motifs in human and rat titin N2-Bus. Phosphosite-specific antibodies were generated against pSer4010, pSer4099, pSer4185 in the N2-Bus, and pSer11878 in the PEVK region. Titin phosphorylation status was determined by Western blot analysis in a set of human donor and failing hearts selected for similar titin isoform composition. In N2B-titin from failing hearts, relative N2-Bus phosphorylation of all three investigated phosphosites was significantly reduced by >28%, whereas relative PEVK phosphorylation was increased by 23% compared to donor hearts. Passive stiffness was analyzed using skinned fiber preparations, and was significantly increased in fibers from failing hearts compared to controls. Our data suggest that the observed differential phosphorylation of the N2B and PEVK region of titin acts complementary to increase passive tension in the myocardium, and that titin phosphorylation is an important physiological mechanism to fine-tune passive myocardial stiffness, and thus diastolic function of the heart.