The aim of this study is to investigate the effect and mechanism of 17 beta-estradiol (E2) on oxidative stress in theosteoblasts. An oxidative stress-induced damage model was established using H2O2 in MC3T3-E1 cells, andH2O2-induced cells were treated with E2. The results indicated that E2 attenuated oxidative stress in H2O2-induced MC3T3-E1 cells. In addition, H2O2 upregulated the expression of miR-320-3p and downregulated thatof RUNX2, but these changes were counteracted by E2. Thereafter, we verified the interactive relationshipbetween miR-320-3p and RUNX2. Then, H2O2-induced MC3T3-E1 cells were transfected with miR-320-3pmimics or inhibitors and treated with E2. The results indicated that miR-320-3p inhibition suppressed H2O2-induced inflammation, apoptosis, and oxidative stress and promoted the osteogenic differentiation of MC3T3-E1 cells by regulating RUNX2, ALP, and OCN, and this effect was further strengthened by E2. In conclusion,the findings suggest that E2 alleviates oxidative damage in osteoblasts by regulating the miR-320/RUNX2signaling.