There are controversial findings regarding the roles of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway on bone metabolism under oxidative stress. We investigated how Nrf2/HO-1 pathway affects osteoblast differentiation of MC3T3-E1 cells in response to hydrogen peroxide (H 2O 2), N-acetyl cysteine (NAC), or both. Exposing the cells to H 2O 2 decreased the alkaline phosphatase activity, calcium accumulation, and expression of osteoblast markers, such as osteocalcin and runt-related transcription factor-2. In contrast, H 2O 2 treatment increased the expression of Nrf2 and HO-1 in the cells. Treatment with hemin, a chemical HO-1 inducer, mimicked the inhibitory effect of H 2O 2 on osteoblast differentiation by increasing the HO-1 expression and decreasing the osteogenic marker genes. Pretreatment with NAC restored all changes induced by H 2O 2 to near normal levels in the cells. Collectively, our findings suggest that H 2O 2-mediated activation of Nrf2/HO-1 pathway negatively regulates the osteoblast differentiation, which is inhibited by NAC. [BMB Reports 2015; 48(11): 636-641]