BACKGROUND: In applications on dental/orthopedic implants and bone regeneration, biomaterials contained magnesium have been widely used. However, the mechanism underlying the biologic effects is still largely unknown. In addition, previous reports of osteogenic effect of magnesium mainly relied on studies using ATCC osteosarcoma cell lines but not normal human osteoblasts. OBJECTIVE: This study was designed to test the effect of magnesium on osteogenic phenotypic behaviors of normal human osteoblasts. METHODS: Normal human osteoblasts derived from human alveolar bone were cultured in triplicate in growth media with varies concentrations of supplemental magnesium: 0.5mM, 1mM, 2mM, 4mM, 8mM and 16mM as the study groups and 0mM as a control group for the time intervals of 7 days, 10 days, 14 days and 21days. Cell proliferation was measured by crystal violet dye staining. Expression of osteocalcin was measured by Quantikine Elisa and mineralization of cultures was measured by Alizarin Red staining. The data were normalized per cell basis. Statistical analysis was done using ANOVA and Student’s t test. Results: Osteocalcin expression was upregulated in groups with supplemented magnesium at 0.5mM (1.16folds, p<0.01 ), 1.0mM (1.22folds, p<0.01 ), 2.0mM (1.37folds, p<0.01 ) at day 21 compared to control, while at 4mM ( p<0.01 ) and above showed down-regulation. Alizarin Red stained cultures showed higher degree of mineralization at 1mM ( p=0.0228 ) and 2mM ( p=0.0142) compared to control. Groups with 4mM and above showed less calcium deposition. Similar results have been gained also on day 10 and day 14 for both assays. CONCLUSION: Osteogenesis of normal human osteoblasts could be significantly upregulated by 2mM supplemental magnesium. These data are important for manufacturing magnesium-containing biomaterials for bone tissue regeneration and implants.