Objective To explore the protective effect and associated mechanism of action of estrogen on oxidative stress injury of mouse MC3T3-E1 osteoblasts (OB). Methods MC3T3-E1 cells cultured in vitro were divided into a blank control group, H₂O₂ group (300 μmol / L), H₂O₂+ 0. 1 μmol / L estrogen group, H₂O₂+ 1 μmol / L estrogen group, H₂O₂+ 10 μmol / L estrogen group, and H₂O₂+ 1 mmol / L NAC group, which were incubated with the corresponding concentrations of the drugs. The proliferative activities of cells in each group were determined by CCK-8. The apoptosis rate and mitochondrial membrane potential (MMP) were determined by flow cytometry. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were determined using kits. The level of reactive oxygen species (ROS) was determined by a fluorescent probe method . The expression of Smad5, Runx2, Bax, Bcl-2, and cleaved Caspase-3 proteins was determined by western blotting. Results Compared with those in the blank control group, the proliferative activities of cells were significantly decreased (P< 0. 05), apoptosis rate and positive rate of JC-1 cells were significantly increased (P< 0. 05), levels of MDA and ROS were significantly increased (P< 0. 05), SOD activity was significantly decreased (P< 0. 05), expression of Smad5, Runx2, and Bcl-2 proteins was significantly decreased ( P < 0. 05), and expression of Bax and cleaved Caspase-3 proteins was significantly increased in the H₂O₂ group (P< 0. 05). Compared with those in the H₂O₂ group, proliferative activities of cells were significantly increased (P< 0. 05), apoptosis rate and positive rate of JC-1 cells were significantly decreased (P< 0. 05), levels of MDA and ROS were significantly decreased (P< 0. 05), SOD activity was significantly increased (P< 0. 05), expression of Smad5, Runx2, and Bcl-2 proteins was significantly increased (P< 0. 05), and expression of Bax and cleaved Caspase-3 proteins was significantly decreased in the H₂O₂+ 1 μmol / L estrogen group, H₂O₂+ 10 μmol / L estrogen group, and H₂O₂ + 1 mmol / L NAC group (P< 0. 05). Conclusions Estrogen may reduce the level of ROS in MC3T3-E1 cells damaged by oxidative stress and improve the differentiation ability of cells by activating the expression of the Smad5 / Runx2 signal axis.