BACKGROUND: Reactive oxygen species play critical roles in homeostasis and cell signaling. Dexmedetomidine, a specific agonist of the α₂-adrenoceptor, has been commonly used for sedation, and it has been reported to have a protective effect against oxidative stress. In this study, we investigated whether dexmedetomidine has a protective effect against H₂O₂-induced oxidative stress and the mechanism of H₂O₂-induced cell death in normal human fetal osteoblast (hFOB) cells. METHODS: Cells were divided into three groups: control group—cells were incubated in normoxia without dexmedetomidine, hydrogen peroxide (H2O2) group—cells were exposed to H₂O₂ (200 µM) for 2 h, and Dex/H₂O₂ group—cells were pretreated with dexmedetomidine (5 µM) for 2 h then exposed to H₂O₂ (200 µM) for 2 h. Cell viability and apoptosis were evaluated. Osteoblast maturation was determined by assaying bone nodular mineralization. Expression levels of bone-related proteins were determined by western blot. RESULTS: Cell viability was significantly decreased in the H₂O₂ group compared with the control group, and this effect was improved by dexmedetomidine. The Hoechst 33342 and Annexin-V FITC/PI staining revealed that dexmedetomidine effectively decreased H₂O₂-induced hFOB cell apoptosis. Dexmedetomidine enhanced the mineralization of hFOB cells when compared to the H₂O₂ group. In western blot analysis, bone-related protein was increased in the Dex/H₂O₂ group. CONCLUSIONS: We demonstrated the potential therapeutic value of dexmedetomidine in H₂O₂-induced oxidative stress by inhibiting apoptosis and enhancing osteoblast activity. Additionally, the current investigation could be evidence to support the antioxidant potential of dexmedetomidine in vitro.