The precise cellular origin and regulatory mechanisms underlying cementum development remain poorly understood, hindering progress toward ideal cementum regeneration. Bone morphogenetic protein 2 (BMP2), approved by the FDA for clinical use due to its potent osteoinductive capacity, is a key candidate for such regulation. Activin receptor-like kinase 3 (Alk3)-mediated BMP signaling plays a crucial role in the development and structural maintenance of mineralized tissues, including teeth. However, the precise mechanism by which BMP signaling regulates periodontal tissue development mediated by periodontal ligament stem cells (PDLSCs), especially Gli1+ cells, remains unknown. Emerging evidence has indicated that O-GlcNAc glycosylation (O-GlcNAcylation), a dynamic post-translational modification, modulates critical biological processes, such as transcription, translation, and cell fate determination. In this study, we demonstrate how BMP2 signaling enhances O-GlcNAcylation in a SMAD-dependent manner. Notably, we demonstrate that the lack of Alk3 in Gli1+ cells resulted in reduced O-GlcNAcylation levels in vivo. Nonetheless, O-GlcNAcylation is identified as indispensable for PDLSC-mediated osteogenesis and cementogenesis both in vivo and in vitro. Moreover, deleting O-β-N-acetylglucosaminyltransferase (Ogt) in Gli1+ cells suppresses BMP signaling, consequently impairing cellular cementum formation and delaying alveolar socket healing. Mechanistically, we further revealed that the cytoskeleton, especially the MYH9 (non-muscle myosin IIa, NMIIA), is O-GlcNAcylated and is essential for BMP2-induced osteogenesis and cementogenesis. These findings demonstrate the importance of O-GlcNAcylation in periodontal ligament stem cells (PDLSCs) differentiation and provide a promising therapeutic target for alveolar bone regeneration.