In order to investigate the possible role of mesenchyme forkhead-1 (MFH-1) in osteogenesis and osteoblast differentiation, the gene-recombination and hybridization methods are used to produce anti-mouse MFH-1 monoclonal antibody. The expression of MFH-1 induced by bone morphogenetic protein-2 (BMP-2) in myoblasts C2C12 was examined by Western blot and Northern blot analysis. The alkaline phosphatase (ALP) activity and osteocalcin were used as the markers of the osteoblasts lineage, and also were measured. The results showed that the anti-mouse MFH-1 monoclonal antibody was able to identify specifically the mouse MFH-1 protein which was expressed in human bladder carcinoma HTB 9 cell transfected with CX-MFH-1 plasmid by Western blot analysis. The myoblasts C2C12 could express the endogenous MFH-1 protein in its nucleus. MFH-1 protein and MFH-1 mRNA both increased markedly in C2C12 cells after treatment with BMP-2; after lowering the endogenous MFH-1 level by stably transfecting C2C12 cells with antisense MFH-1 sequence, the alkaline phosphatase(ALP) activity and production of osteocalcin induced by BMP-2 were significantly lowered in antisense MFH-1 cell lines than in control cell lines. It can be concluded that the results suggest that the BMP-2-induced MFH-1 protein may play an essential role in regulating the osteoblastic differentiation of myoblasts C2C12.