Estradiol (E2) serves an important role in the changes of postmenopausal bone turnover rate and the development of osteoporosis. The present study aimed to investigate the effects of E2 on high glucose (HG)-induced osteoblast injury. Cell Counting Kit-8 was used to determine cell viability. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting was used to analyze the mRNA and protein expression levels of osteocalcin, Runt-related transcription factor 2 (Runx2), nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO1). Flow cytometry was performed to analyze apoptosis. The results revealed that cell viability was lower in cells treated with HG (100, 200 or 300 mg/dl) compared with the control group. Cell viability was decreased in cells treated with 200 mg/dl HG on days 3, 5 and 7. In addition, cell viability was increased by 0.1 µM E2. E2 with HG co-treatment increased cell viability, osteocalcin and Runx2 mRNA expression levels and nuclear Nrf2 and HO1 protein expression levels compared with the HG-only group. All these changes, with the exception of Runx2, were reversed by silencing Nrf2 expression using small interfering (si)RNA (siNrf2). Additionally, apoptosis was reduced by E2 in HG-treated cells, which was reversed by siNrf2 transfection. These results demonstrated that E2 may prevent HG-induced osteoblast injury by activating Nrf2/HO1 signaling pathways.