Mesenchyme forkhead‐1 (MFH‐1), a winged helix/forkhead transcription factor, is expressed in developing cartilaginous tissues, kidney and arch arteries, and is essential for the normal development of the axial skeleton and aortic arch formation of mice. To investigate the possible role of MFH‐1 in osteogenesis and osteoblast differentiation, we examined expression of MFH‐1 induced by bone morphogenetic protein‐2 (BMP‐2) in C2C12 myoblasts, and found that MFH‐1 protein and also MFH‐1 mRNA increased markedly in C2C12 cells after treatment with BMP‐2. To confirm the hypothesis that BMP‐2 induced osteoblastic differentiation of C2C12 cells by increasing MFH‐1 expression, we lowered the endogenous MFH‐1 level by stably transfecting C2C12 cells with antisense MFH‐1 sequence, and found that in antisense MFH‐1 cell lines, both alkaline phosphatase (ALP) activity and production of osteocalcin induced by BMP‐2 decreased markedly in comparison with control cell lines. Our results suggest that the BMP‐2‐induced MFH‐1 protein may play a key role in regulating the commitment to osteoblastic differentiation of C2C12 myoblasts and production of osteoblast markers including ALP and osteocalcin.