Cardiac titin is the main determinant of sarcomere stiffness during diastolic relaxation. To explore whether titin stiffness affects the kinetics of cardiac myofibrillar contraction and relaxation, we used subcellular myofibrils from left ventricles of homozygous (KO) and heterozygous (HET) N2B-KO mice which express truncated cardiac titins lacking the unique elastic N2B region. Compared to myofibrils from wildtype (WT) mice, myofibrils from KO and HET mice exhibit increased passive myofibrillar stiffness. To determine the kinetics of Ca2+-induced force development (rate constant kACT), myofibrils from KO, HET and WT mice were stretched to the same sarcomere length (2.3 µm) and rapidly Ca2+ activated. Additionally, mechanically-induced force redevelopment kinetics (rate constant kTR) was determined by slackening and re-stretching myofibrils during Ca2+ activation. Myofibrils from KO mice exhibited significant higher kACT, kTR and maximum Ca2+ activated tension than myofibrils from WT. In contrast, the kinetic parameters of biphasic force relaxation induced by rapidly reducing [Ca2+] were not significantly different among the three genotypes. These results indicate that increased titin stiffness promotes myocardial contraction by accelerating the formation of force-generating cross-bridges without decelerating relaxation.