Nuclear factor E2-related factor 2 (nrf2) is accepted as an important transcription factor in the regulation of many phase II detoxifying enzymes and oxidative-stress-inducible genes in many tissues including bone and cartilage. However, little attention has been paid to precise role of nrf2 on osteoblasts and chondrocytes. In the present study, we therefore investigated the role of nrf2 in the regulation of cell proliferation, differentiation and maturation using both MC3T3-E1 and ATDC5 cells. In MC3T3-E1 cells stably transfected with nrf2 (MC3T3-E1-nrf2 clone), the differentiation-dependent induction of alkaline phosphatase activity was significantly inhibited in addition to decrease of the mineralized matrix formation. Moreover, expression of mRNAs for osteocalcin and type I collagen was also markedly attenuated in MC3T3E1-nrf2 clone but not in MC3T3E1-empty vector clone. These inhibitory effect on cell differentiation was also observed in ATDC5 cells stably transfected with nrf2. Moreover, nrf2 significantly impaired the runx2-dependent enhancement of osteocalcin promoter activity. These data suggest that nrf2 may negatively regulate the differentiation of both osteoblasts and chondrocytes through inhibition of the runx2 dependent transcriptional activity.