TNNI3K is a new cardiac-specific MAP kinase whose gene is localized to 1p31.1 and that belongs to a tyrosine kinase-like branch in the kinase tree of the human genome. In the present study we investigate the role of TNNI3K in cardiac myogenesis process and in repair of ischemic injury. Pluripotent P19CL6 cells with or without transfected by pcDNA6-TNNI3K plasmid were used to induce differentiation into beating cardiomyocytes. TNNI3K promoted differentiation process judging from increased number of beating mass and -actinin-positive cells. TNNI3K improved cardiac function by enhancing beating frequency, increasing contractile force and epinephrine response of spontaneous action potentials without increase of the single cell size. TNNI3K suppressed phosphorylation of cardiac troponin I, annexin-V⁺ cells, Bax-protein and p38-, JNK-mediated apoptosis. Intra-myocardial administration of TNNI3K-overexpressing P19CL6 cells in mice with myocardial infarction improved cardiac performance and attenuated ventricular remodeling when compared with injection of wild-type P19CL6 cells. In conclusion, our study clearly indicates that TNNI3K promotes cardiomyogenesis, enhances the cardiac performance and protects the myocardium from ischemic injury through suppressing the p38, JNK-mediated apoptosis. Therefore, modulation of TNNI3K activity would be a useful therapeutic approach for ischemic cardiac disease.