Oxidative stress due to excessive reactive oxygen species (ROS) and depleted antioxidants such as glutathione (GSH) can give rise to apoptotic cell death in acutely diabetic hearts, leading to heart disease. At present, the source of this cardiac ROS or the sub cellular site of cardiac GSH loss [i.e. cytosolic (cGSH) or mitochondrial GSH (mGSH)] has not been completely elucidated. Using rotenone (an inhibitor of the electron transport chain) to decrease the excessive ROS in acute streptozotocin (STZ)-induced-diabetic rat heart, the mitochondrial origin of ROS was established. Further, mitochondrial damage, as evidenced by a loss of membrane potential, increase in oxidative stress and reduction in mGSH was associated with increased apoptosis via increases in caspase-9 and 3 activities in acutely diabetic hearts. To validate the role of mGSH in regulating cardiac apoptosis, buthionine-l-sulfoximine (BSO; 10 mmol/kg, i.p.) [blocks GSH synthesis] or diethyl maleate (DEM; 4 mmol/kg, i.p.) [inactivates preformed GSH] were administered in diabetic rats for 4 days following STZ-administration. Although both BSO and DEM lowered cGSH, they were ineffective in reducing mGSH or augmenting cardiomyocyte apoptosis. To circumvent the lack of mGSH depletion, BSO and DEM were co-administered in diabetic rats. In this setting, mitochondrial GSH was undetectable and cardiac apoptosis was further aggravated compared to the untreated diabetic group. In a separate group, GSH supplementation induced a robust amplification in mGSH in diabetic rat hearts and prevented apoptosis. Our data for the first time suggests that mGSH is crucial for modulating the cell suicide program in short-term diabetic rat hearts.