Grape seed proanthocyanidin extract (GSPE), a polyphenolic compound with antioxidant properties, may protect against cardiac ischemia and reperfusion injury. However, its potential toxicity at higher doses is unknown. The authors tested the effects of GSPE on reactive oxygen species (ROS) generation, cell survival, lactate dehydrogenase (LDH) release, and caspase-3 activity using chick cardiomyocytes incubated with GSPE at 5, 10, 50, 100, or 500 μg/mL in medium for 8 h. Exposure to increasing concentrations of GSPE (100 or 500 μg/mL) resulted in an increase in ROS generation and cell death as measured by propidium iodide uptake and LDH release. Caspase-3 activity was significantly increased fourfold in cells exposed to GSPE 500 μg/mL compared to controls; this was abolished by the selective caspase-3 inhibitor AC-Asp-Gln-Thr-Asp-H(50 μM), which aslso significantly reduced the cell death resulting from GSPE (500 μg/mL). The antioxidant N-acetylcysteine (NAC, 100 μM) reduced cell death induced by GSPE (500 μg/mL) but failed to attenuate caspase-3 activation. Collectively, the authors conclude that higher doses of GSPE could cause apoptotic cell injury via effector caspase-3 activation and subsequent induction of ROS generation. Consumers may take higher doses of dietary supplements in the belief that natural herbs have no major side effects. This study demonstrates that dosages of GSPE should be optimized to avoid potential harmful pro-oxidant effects. Terry L. Vanden Hoek and Zuo-Hui Shao are equal contributors to this work.