Mitochondrial transcription factor A (TFAM) is essential for mitochondrial DNA transcription and replication. TFAM transcriptional activity is decreased in diabetic cardiomyopathy, however, the functional implications are unknown. We hypothesized that a reduced TFAM activity may be responsible for some of the alterations caused by hyperglycemia. Therefore, we investigated the effect of TFAM over-expression on hyperglycemia-induced cytosolic calcium handling and mitochondrial abnormalities. Neonatal rat cardiomyocytes were exposed to high glucose (30mM) for 48 h and we examined whether TFAM over-expression, by protecting mitochondrial DNA, could reestablish calcium fluxes and mitochondrial alterations towards normal. Our results shown that TFAM over-expression increased to more than two fold mitochondria copy number in cells treated either with normal (5.5mM) or high glucose. ATP content was reduced by 30% and mitochondrial calcium decreased by 40% after high glucose. TFAM over-expression returned these parameters to even higher than control values. Calcium transients were prolonged by 70% after high glucose which was associated with diminished SERCA2a and cytochrome c oxidase subunit 1 expression. These parameters were returned to control values after TFAM over-expression. High glucose-induced protein oxidation was reduced by TFAM over-expression indicating a reduction of the high glucose-induced oxidative stress. In addition, we found that TFAM activity can be modulated by O-linked -N-acetylglucosamine glycosylation. In conclusion, TFAM over-expression protected cell function against the damage induced by high glucose in cardiomyocytes.