<p>[Background] Honokiol is a small-molecule polyphenol isolated from the genus Magnolia and known as an activator of sirtuin 3, a mitochondrial deacetylase.  We examined whether honokiol attenuates mitochondrial injury, leading to the attenuation of cell death in hypoxia-reoxygenation (H/R) injury in cardiomyocytes. [Methods and Results] Neonatal rat cultured cardiomyocytes were subjected to 5 hours of hypoxia followed by 30 minutes of reoxygenation in the presence or absence of honokiol (30 μmol/L). Lethal myocyte injury was assessed by LDH activity in culture medium and myocyte apoptosis was examined by nuclear staining with DAPI and caspase 3 activity. H/R significantly increased LDH activity and apoptotic myocytes, and treatment with honokiol significantly attenuated these indices of myocyte death.  In mitochondrial apoptotic pathway, reduction of mitochondrial membrane potential plays critical roles, and ATP is mainly produced by mitochondria.  After H/R mitochondria lost their membrane potential, detected by TMRM fluorescence, leading to reduction of ATP content in myocytes, and honokiol recovered them. After H/R protein expression of sirtuin 3 was significantly restored by honokiol.  Sirtuin 3 is known to deacetylate Mn-SOD. After H/R honokiol decreased acetylation levels of Mn-SOD and tended to attenuate mitochondrial hydrogen peroxide production.  [Conclusion] These results indicate that Honokiol protects mitochondria via enhancement of sirtuin 3, leading to attenuation of H/R-induced myocyte lethal injury.</p>