Tanshinone I, a naturally occurring diterpene from Danshen, has been shown to possess hepatocyte protective, anticancer, and memory enhancing properties. However, there are few stringent pharmacological tests for neuroprotection of tanshinone I thus far. Since peroxynitrite is involved in the pathogenesis of neurodegenerative disorders, this study was undertaken to investigate whether the neuroprotective effect of tanshinone I is associated with inhibition of peroxynitrite-caused DNA damage, a critical event leading to peroxynitrite-induced cytotoxicity. Our results show that tanshinone I can significantly inhibit peroxynitrite-induced DNA damage both in φX-174 plasmid DNA and rat primary astrocytes. EPR spectroscopy indicates that tanshinone I potently diminished the DMPO-hydroxyl radical adduct signal from peroxynitrite. Taken together, these results demonstrate for the first time that tanshinone I can protect against peroxynitrite-induced DNA damage, hydroxyl radical formation and cytotoxicity, which might have implications for tanshinone I-mediated neuroprotection.