Due to a limited ability to achieve self-repair and regeneration as well as lack of effective therapeutic options, degeneration and injury of the pulp-dentin complex may result in severe consequences. Magnesium-based biomaterials might provide an innovative therapeutic potential to substantially enhance regeneration of dental tissues. Magnesium (Mg 2+ ) has been considered for its potential ability to accelerate proliferation and differentiation of human osteoblasts. However, to date, magnesium oxide (MgO) and its dentinogenic effects on human dental pulp cells (HDPCs) has not been investigated. This study was designed to evaluate the stimulatory effect of different concentrations of MgO on dentinogenesis of HDPCs. HDPCs were cultured with 0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM concentrations of supplemental MgO, 0 mM as the negative control group, lignin sulfonic acid sodium salt and xanthan gum as the vehicle control groups. Cell attachment efficiency was assessed at 16 h. Proliferation rate was evaluated at 3, 7, 10, 14 days. Both attachment efficiency and proliferation rate were assessed by crystal violet staining. Cell viability was determined by activity of mitochondrial dehydrogenase enzyme. Alkaline phosphatase (ALP) activity was assessed using fluorometric assay at 7, 10, and 14 days. Mineralization of cultures was measured by Alizarin Red staining. Statistical analysis was performed using multi-way ANOVA with Wilks' lambda test. Higher cell attachment efficiency was shown with 0.5 mM at 16 hours compared to negative control (P<0.001). Cells with 0.5 mM supplemental MgO showed significantly higher proliferation rates than negative and vehicle controls at 7, 10, and 14 days (P<0.001). Higher levels of ALP activity and mineralization were also observed in 0.5 mM supplemental MgO at 10, and 14 days (P<0.001). In conclusion, optimal MgO (0.5 mM) group significantly upregulated HDPCs attachment, proliferation, ALP activity, odontogenic differentiation and mineralization. Magnesium oxide containing biomaterials could be a potential novel material for pulp and dentin repair in regenerative endodontics.