Troponin T (TnT) is a striated muscle-specific protein and an abundant component of the myofilaments. Non-myofilament-associated TnT is rapidly degraded in myocytes, implying an importance in the maintenance of cellular environment. However, if the level of non-myofilament-associated TnT or TnT fragments exceeds the degradation capacity, it may cause cytotoxicity. To investigate this hypothesis, we constructed bi-cistronic vectors to express different portions of TnT polypeptide chain together with non-fusion green fluorescent protein as a tracer for the transfection. Cytotoxicity of the TnT fragments was studied through forced expression in C₂C₁₂ myoblasts and HEK293 non-muscle cells and the viability of the transfected cells was examined. The results demonstrated that in the absence of myofilaments, the conserved C-terminal and middle fragments of TnT were highly effective on inducing cell death via apoptosis whereas the N-terminal variable region was not. As combined effects, non-myofilament-associated intact cardiac TnT and a C-terminal truncated slow TnT fragment found in Amish nemaline myopathy exhibited intermediate cytotoxicity. Considering that peak releases of TnT or TnT fragments from decomposition of a large number of myofibrils in acute myocardial infarction may breach the cellular protection of proteolytic degradation and result in apoptosis as a potential cause for the loss of cardiomyocytes.