Alpha-lipoic acid (LA) as a dietary supplement strongly scavenges free radicals. The negative relationship between oxidative stress and bone formation has recently been reported. Here, we investigated the protective effects of LA on the hydrogen peroxide-induced oxidative stress and impaired calcification in MC3T3-E1 osteoblast-like cells. After exposure to H2O2, there was a decrease in the levels of calcification and reduced glutathione but an increase in thiobarbituric acid-reactive substances levels in the no LA-treated cells. The presence of LA attenuated oxidative stress and significantly enhanced calcification capacity at 0.1 and 1.0 mM (P < 0.05). The quantitative real-time PCR results showed that LA upregulated expression of genes engaged in antioxidant enzymes and mineralization, including catalase, glutathione peroxidase 3, and osteocalcin. Additionally, LA suppressed the over-expression of genes involved in p53 apoptotic pathway, including p53 and caspase 3. These results indicate that LA protects MC3T3-E1 cells against the H2O2-induced oxidative damage to mineralization.Highlights► α-Lipoic acid (LA) prevented the H2O2-induced oxidative stress in MC3T3-E1 cells. ► LA attenuated the impaired calcification induced by H2O2. ► LA upregulated the expression of antioxidant enzymes, including Cat and Gpx3. ► LA increased the mineralization-related genes expression, such as Col1a1 and Oc. ► LA suppressed the p53 apoptotic pathway-related genes expression.