Background <p>Metformin has emerged as a potential bioactive agent in regenerative medicine; however, its optimal concentration for enhancing dental pulp stem cell (DPSC) function remains unclear. This study evaluated the dose-dependent effects of Metformin (500 µM and 1000 µM) on DPSC proliferation and odontogenic differentiation.</p> Methods <p>Cell viability was assessed after 48&#xa0;h, while differentiation was examined using alkaline phosphatase (ALP) activity at days 3 and 7, Alizarin Red S staining for mineralization at days 7 and 14, and gene expression analysis of <i>RUNX2</i>, <i>DMP1</i>, and <i>BMP2</i> at day 14.</p> Results <p>Low-dose Metformin (500 µM) significantly increased cell viability, enhanced ALP activity, promoted mineralized matrix deposition, and upregulated odontogenic gene expression compared to control cultures. In contrast, the higher concentration (1000 µM) attenuated proliferation and differentiation markers, demonstrating a biphasic biological response.</p> Conclusion <p>These findings indicate the presence of a therapeutic window in which Metformin optimally stimulates odontogenic maturation without inducing inhibitory effects. The results provide biologically grounded evidence supporting the future integration of controlled-dose Metformin into pulp capping materials and regenerative endodontic therapies, while emphasizing the necessity of precise dose optimization for clinical translation.</p> Graphical abstract <p></p>