Hydroxyapatite (HA) is perhaps the most attractive material for bone repair, replacement and regeneration, due to its chemical composition and crystallographic structure which are similar to those of natural bone mineral. However, replacement of various elements and compounds in HA, could improve biological properties of this material. The aim of this study was preparation, characterization and bioactivity evaluation of silicon and magnesium co-doped fluorapatite (Si-Mg-FA). Structural characterizations of synthesized powder were performed using X-ray diffraction (XRD) analysis; Fourier transformed infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) techniques. In vitro bioactivity was evaluated in simulated body fluid (SBF) at 37˚C for up to 28 days. Cell viability and cell attachment were studied by MTT assay and scanning electron microscopy (SEM).  The results showed that nanosized (~40nm) single-phase Si-Mg-FA powder was synthesized after 12 h of ball milling. In vitro examinations revealed the amount of bone-like apatite precipitated on Si-Mg-FA nanopowder was significantly higher than FA. The cell culture medium containing Si-Mg-FA showed more cell proliferation and cell viability than FA.  It could be concluded that doping Si and Mg into FA improves the bioactivity and cell viability, therefore, has a good potential to be used as bone substitution material.