High glucose (HG) state is closely related to diabetic complications, and among these one main pathogenesis involves apoptosis of important functional cells. However, the mechanism of osteoblast (OB) apoptosis induced by HG state is not clear. Peroxynitrite (ONOO-), the strongest oxidant, can mediate apoptosis in various kinds of cells, including OB. Therefore, this study was aimed at investigating whether HG state could induce OB apoptosis through ONOO-. Cultured OB from rat calvariae explanted from E21 fetuses were treated with HG solution. The ratio of OB apoptosis and ONOOcontent was assayed by TUNEL, flow cytometry, and immunohistochemistry. The results showed that HG state could induce ONOO- overformation (p<0.01), and excessive OB apoptosis (p<0.001). However, the HG-induced OB apoptosis could be attenuated by peonol, a potent scavenger of ONOO-. In conclusion, HG state might trigger excessive ONOO- formation, mediating OB apoptosis.