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Periodontal defect regeneration in severe periodontitis relies on the differentiation and proliferation of periodontal ligament cells (PDLCs). Recently, an emerging 2D nanomaterial, MXene (Ti3C2Tx), has gained more and more attention due to the extensive antibacterial and anticancer activity, while its potential biomedical application on tissue regeneration remains unclear. Through a combination of experimental and multiscale simulation schemes, Ti3C2Txhas exhibited satisfactory biocompatibility and induced distinguish osteogenic differentiation of human PDLCs (hPDLCs), with upregulated osteogenesis‐related genes. Ti3C2Txmanages to activate the Wnt/β‐catenin signaling pathway by enhancing the Wnt‐Frizzled complex binding, thus stabilizing HIF‐1αand altering metabolic reprogramming into glycolysis. In vivo, hPDLCs pretreated by Ti3C2Txdisplay excellent performance in new bone formation and osteoclast inhibition with enhanced RUNX2, HIF‐1α, and β‐catenin in an experimental rat model of periodontal fenestration defects, indicating that this material has high efficiency of periodontal regeneration promotion. It is demonstrated in this work that Ti3C2Txhas highly efficient therapeutic effects in osteogenic differentiation and periodontal defect repairment. MXene (Ti3C2Tx) enhances human periodontal ligament cells' osteogenetic differentiation in vitro and periodontal regeneration in a rat periodontal defect model by activating WNT/hypoxia‐inducible factors‐1α(HIF‐1α)‐mediated metabolism reprogramming, marked by increasing β‐catenin, HIF‐1α, and succinic acid.