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Purpose: This research to evaluate and compare the effect of Nigella Sativa Oil (NSO) on the stemness properties of human dental pulp stem cells (DPSCs). Materials and methods: (DPSCs) were isolated from the pulp of three permanent teeth indicated for extraction for therapeutic purposes. DPSCs were cultured with NS oil at concentration 10μM. and with CaOH and MTA extracts as positive controls. Cells cultured without any extract were used as negative controls. Stemness properties of DPSCs were assessed terms of proliferation, migration, and the ability to enhance wound healing. Moreover, the osteogenic differentiation potential of DPSCs cultured with NSO was evaluated. Results: No significant statistical difference between groups was detected groups regarding cell proliferation rate. Also, no significant difference between the numbers of migrated DPSCs in the NSO group when compared to those in the –ve control group (p>0.05) while there was a significant difference between the number of migrated DPSCs in the NSO and +ve control groups. The ability to enhance the healing of the wound was higher in the MTA group rather than in the NSO group. Finally, successful osteogenic differentiation of DPSCs manifested as calcified nodules occurred in all groups. Conclusions: Because of the limitation of the current research it concluded that NSO may have the potentials for pulp capping and regenerative applications. Conclusions:Despite the limitations of this study, NSO proved to have no adverse effect on DPSCs proliferation rate and can induce osteogenic differentiation of DPSCs. On the other hand, NSO showed little effect on cell migration and wound healing in DPSCs.