Human periodontal ligament stem cells (hPDLSCs) are ideal seed cells for periodontal regeneration. To better understand the dynamic protein profiles during hPDLSCs osteogenic differentiation, we conducted a quantitative proteomics to reveal the temporal protein expression pattern during this process. Clustering and functional annotation by Gene Ontology terms identified distinctions among proteins with different dynamic tendencies. Pathway enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes database found that oxidative phosphorylation (OXPHOS) was significantly involved, which further predicted continuous activation by Ingenuity Pathway Analysis software. Redox-sensitive signalling pathways with dynamic activation status showed associations with OXPHOS to various degrees, especially the sirtuin signalling pathway. SOD2, an important component of the sirtuin signalling pathway, is a hub protein of the interaction network and displays a persistent increase during osteogenesis. Our in-depth dynamic proteomic analysis indicated the comprehensive and dynamic regulation mechanism during hPDLSC osteogenesis.