Marsilea crenata Presl. leaves contain phytoestrogens that is suspected to play a role in increasing bone formation. Bone formation activity is defined by the expression of Osterix, a transcription factor that plays a key role in bone development. The aim of this study is to show that the ethyl acetate fraction of Marsilea crenata Presl. leaves can increase bone formation process in hFOB 1.19 cells in a TNF-α dependent manner, by measurement of Osterix. The hFOB 1.19 cells were grown in 24-well microplates and treated with 10 ng/ml TNF-α for 24 hours. The ethyl acetate fraction of Marsilea crenata Presl. leaves were also added in concentrations of 62.5, 125, and 250 ppm. A positive control was employed, at a concentration of 2.5 g/L of genistein. The expression of osterix was examined using an immunocytochemistry technique with CLSM to assess bone forming activity. The results show that ethyl acetate fraction Marsilea crenata Presl. leaves can boost osterix expression in hFOB 1.19 cells, with optimal concentration was 250 ppm at p<0.005. The ethyl acetate fraction of Marsilea crenata Presl. leaves can increase Osterix in osteoblast hFOB 1.19 cell, indicating improved bone forming activity.