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Objectives: The aim of this study was to assess the cytotoxic effects and osteogenic activity of recombinant human bone morphogenetic protein (rhBMP2) and nano-hydroxyapatite (n- HA) adjacent to MG-63 cell line. Materials and Methods: To assess cytotoxicity, the 4,5-dimethyl thiazolyl-2,5-diphenyl tetrazolium bromide (MTT) assay was used. Alkaline phosphatase (ALP) activity and oste- ogenic activity were evaluated using Alizarin red and the von Kossa staining and analyzed by one-way ANOVA followed by Tukey’s post hoc test. Results: The n-HA/CS mixture significantly promoted cell growth in comparison to pure calcium sulfate (CS). Moreover, addition of rhBMP2 to CS (P=0.02) and also mixing CS with n-HA led to further increase in extracellular calcium production and ALP activity (P=0.03). Conclusion: This in vitro study indicates that a scaffold material in combination with an osteoinductive material is effective for bone matrix formation.