Periodontal ligament stem cells (hPDLSCs) are mechanosensing cells responding to mechanical forces. This study investigates the impact of decellularised extracellular matrix (dECM) derived from intermittent compressive force (ICF)-treated hPDLSCs on osteogenic differentiation. hPDSCLs were subjected to ICF loading at 1.5 g/cm for 24 h and then maintained with normal medium (N) or osteogenic induction medium (OM) followed by a decellularisation process. dECMs derived from ICF (dECM-ICF) were characterised using a scanning electron microscope, energy-dispersive X-ray spectroscopy, and proteomic analysis. hPDLSCs were re-seeded on dECM-ICF. Cell proliferation and viability were examined by resazurin and LIVE/DEAD assays. Mineralisation was determined by Alizarin Red S staining. Results demonstrated that dECM-ICF-derived from OM (dECM-ICF-OM) exhibited a fibrillar network structure and showed no cellular component while preserving fibronectin and type I collagen. dECM-ICF exhibited biocompatibility, as indicated by the absence of cytotoxic effects and the ability of hPDLSCs to attach, spread, and proliferate. dECM-ICF-OM significantly enhanced mineral deposition compared to dECM-ICF from normal conditions (dECM-ICF-N). Proteomic analysis of dECM-ICF demonstrated the upregulated proteins in the PI3K-Akt, Ras, MAPK, mTOR, ErbB, TNF, and VEGF signallings. In conclusion, dECM-ICF supports hPDLSCs growth and osteogenic differentiation. dECM-ICF is a promising cell-free natural scaffold to promote periodontal regeneration.