Background <p>The dentin-pulp complex (DPC) is composed of the odontoblastic layer and associated stromal components. It serves key functions in immunological homeostasis and tissue regeneration of dental tissues. Human dental pulp stem cells (hDPSCs) have emerged as pivotal cells for DPC regeneration. Current research frontiers primarily focus on developing novel strategies to increase the odontogenic differentiation potential and regenerative efficacy of hDPSCs. This study aims to boost the capacity of hDPSCs to regenerate DPC through mitochondrial transplantation.</p> Methods <p>Mitochondria were isolated from donor hDPSCs and transplanted into recipient hDPSCs (Mito-hDPSCs) in the same passage. Subsequently, cell viability and mitochondrial transplantation efficiency were evaluated <i>via</i> CCK-8, β-galactosidase staining, mitochondrial imaging, and flow cytometry. Furthermore, Mito-hDPSCs’ metabolic capacity was assessed by mitochondrial membrane potential assays and cellular oxidative phosphorylation assays. Moreover, Alkaline Phosphatase (ALP) activity, Alizarin Red S (ARS) staining, RT-qPCR, and Western blotting (WB) were performed to assess Mito-hDPSC’s odontogenic differentiation potential. Moreover, a nude mouse model was used to assess how Mito-hDPSCs induce DPC regeneration in vivo. RNA-Seq analysis was conducted to examine the expression of signaling pathways in Mito-hDPSCs. In addition, ALP, ARS, WB, and Ca²⁺ fluorescence staining were carried out to analyze the underlying mechanisms between mitochondria and the Ca²⁺/Transcription factor activating protein 2α (TFAP2A) signaling axis.</p> Results <p>The results revealed that mitochondrial transplantation enhanced the viability of Mito-hDPSCs. Furthermore, an increased mitochondrial transplant rate was observed at a recipient-to-donor cell ratio of 1:3. Moreover, Mito-hDPSCs demonstrated increased odontogenic differentiation and formed more dentin-pulp-like tissue in vivo. Ca²⁺ signaling and odontogenesis were significantly enriched in Mito-hDPSCs. TFAP2A was identified as a key transcription factor in the odontogenic differentiation of Mito-hDPSCs. Knockdown array revealed that mitochondrial transplantation effectively upregulated TFAP2A expression in Mito-hDPSCs. Furthermore, mitochondrial transplantation elevated intracellular Ca²⁺ concentration, which in turn increased TFAP2A expression.</p> Conclusions <p>Mitochondrial transplantation may promote DPC regeneration by regulating the Ca²⁺/TFAP2A signaling axis in Mito-hDPSCs.</p> Graphical Abstract <p></p>