Background <p>Dental pulp stem cells (DPSCs) aging impedes its application in tooth regeneration techniques, involving abnormal mitophagy. O-GlcNAcylation is a post-translational modification that regulates various cellular processes. Here, we aimed to investigate the role of O-GlcNAcylation in mitophagy and senescence.</p> Methods <p>DPSCs were cultured and passaged in vitro, and the 7th (p7) and 15th (p15) generation cells were collected. OGA and KLF2 were knocked down in p15 cells. Cell senescence was evaluated using senescence associated β-galactosidase staining, enzyme-linked immunosorbent assay, and western blotting; mitophagy was evaluated using western blotting. The regulation of OGA on the O-GlcNAcylation of KLF2 was analyzed using immunoprecipitation and western blotting.</p> Results <p>The results showed that p15 cells were more senescent than p7 cells and had poor mitophagy, with the higher expression of OGA. Knockdown of OGA inhibited senescence and promoted mitophagy in DPSCs. Moreover, silencing of KLF2 reversed the effects on senescence and mitophagy mediated by OGA knockdown. Additionally, OGA suppressed the O-GlcNAcylation of KLF2 at S177 site and thus reduced its stability.</p> Conclusion <p>Silencing of OGA promotes mitophagy and inhibits DPSC senescence by promoting the O-GlcNAcylation of KLF2, suggesting a novel mechanism underlying DPSC senescence.</p>