Background <p>Salvianolic acid B (Sal B), a natural polyphenol with potential therapeutic applications, has been reported to induce reactive oxygen species (ROS) generation. However, its underlying mechanism has not yet been fully elucidated. In this study, we investigated copper-mediated oxidative DNA damage induced by Sal B.</p> Results <p>Sal B significantly increased the level of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in HL-60 cells, but not in H₂O₂-resistant HP100 cells. The formation of 8-oxodG was inhibited by a Cu(I)-specific chelator. These results suggested that Cu(I) and H₂O₂ play critical roles in this process. In calf thymus DNA, Sal B induced 8-oxodG formation in the presence of Cu(II), which was markedly enhanced in the presence of NADH. Using ³²P-5′-end-labeled DNA fragments, we showed that treatment with Sal B in combination with Cu(II) and NADH caused DNA strand breaks and site-specific base modification, especially at thymine and cytosine residues. These results suggest the involvement of ROS other than •OH and this was further supported by radical scavenger experiments. Furthermore, theoretical calculation data suggest that one of the catechol groups in Sal B is electron-donating. Collectively, these results indicate that Cu(II)-mediated autoxidation of the catechol group in Sal B generates Cu(I) and H₂O₂, likely leading to a Cu(I)-hydroperoxide complex formation and resultant oxidative DNA damage. NADH enhances ROS production and oxidative DNA damage by reducing oxidized Sal B and promoting its recycling.</p> Conclusions <p>The potential pro-oxidant risk of Sal B should be carefully evaluated when used as a therapeutic agent.</p>