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 overexpression on hyperglycemia-induced cytosolic calcium handling and mitochondrial abnormalities. Neonatal rat cardiomyocytes were exposed to high glucose (30 mM) for 48 h, and we examined whether TFAM overexpression, by protecting mitochondrial DNA, could reestablish calcium fluxes and mitochondrial alterations toward normal. Our results shown that TFAM overexpression increased to more than twofold mitochondria copy number in cells treated either with normal (5.5 mM) or high glucose. ATP content was reduced by 30% and mitochondrial calcium decreased by 40% after high glucose. TFAM overexpression returned these parameters to even higher than control values. Calcium transients were prolonged by 70% after high glucose, which was associated with diminished sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a and cytochrome-c oxidase subunit 1 expression. These parameters were returned to control values after TFAM overexpression. High glucose-induced protein oxidation was reduced by TFAM overexpression, indicating a reduction of the high glucose-induced oxidative stress. In addition, we found that TFAM activity can be modulated by O-linked beta-N-acetylglucosamine glycosylation. In conclusion, TFAM overexpression protected cell function against the damage induced by high glucose in cardiomyocytes.