The differentiation of dental papilla cells (DPCs) into functional odontoblasts is critical for dentinogenesis, yet the role of mitochondrial dynamics remains unclear. Here, we investigated the functional role of mitochondrial fission and mitochondria-associated endoplasmic reticulum membranes (MAMs) in the odontogenic differentiation of DPCs. Using in vitro differentiation models combined with confocal microscopy, transmission electron microscopy, and gain- and loss-of-function approaches, we found that odontogenic induction triggered early mitochondrial fragmentation and increased MAM formation. Dynamin-related protein 1 (DRP1) mediated mitochondrial fission, which in turn regulated MAM architecture and promoted differentiation. Malic enzyme 2 (ME2) acted as an upstream regulator, facilitating DRP1 recruitment and organizing MAM integrity. Notably, disruption of the ME2-DRP1-MAM axis impaired dentin formation both in vitro and in vivo, either by ME2 knockdown or pharmacological inhibition of DRP1 (Mdivi-1). These findings establish the ME2-DRP1-MAM axis as a critical metabolic–organellar switch driving odontoblast differentiation, providing new mechanistic insights into dentinogenesis and identifying potential therapeutic targets for dentin–pulp complex regeneration.

2-deoxyglucose (2-DG), targeting Hexokinase I (HKI) to inhibit glycolysis in cancer cells, has inspired drug design. Increasing evidence suggests that its phosphorylated form, 2-deoxyglucose-6-phosphate (2-DG-6-P), may inhibit HKI more effectively, highlighting the need to understand the molecular mechanisms behind this effect. Using molecular simulations, we examined interactions between HKI and its substrates—glucose (G) and glucose-6-phosphate (G-6-P)—and inhibitors—2-DG and 2-DG-6-P. G and G-6-P showed stable interactions with HKI, involving residues like ILE 662, SER 588, and PRO 590, while 2-DG had less stable interactions. However, 2-DG-6-P formed the most stable complex, engaging additional residues such as VAL 663 and THR 848, with enhanced binding through hydrophobic and hydrogen bond interactions facilitated by its phosphorylated group. This study reveals the differential effects of 2-DG and 2-DG-6-P on HKI inhibition, highlighting the unique mechanism of 2-DG post-phosphorylation and supporting the development of targeted HKI inhibitors that exploit cancer cell metabolism.