Abstract: Objective To obtain regulating proteins during human periodontal ligament cell (hPDLC) nstengenic differentiation and investigate its underlying molecular mechanism. Methods Two-dimension difference in gel electrophoresis (2D-DIGE) coupled with matrix-assissted laser adsorption ionization-time of flight mass spectrometry ( MALDI-TOF-MS ) analysis was used to identify regulating proteins during hPDLC osteogenic differentiation. Results A differently expressed protein profile was obtained 7 days after osteogenic induction, and 61 protein spots with significant differences, 29 protein spots were identified by MALDI-TOF-MS, including cytoskeleton, cell membrane-bounding, nuclear regulation,matrix synthesis, and metabolic enzymes and signal tnmsduction and other proteins. A functional class of cyteskeleton proteins showed co-regulation, these proteins were intimately involved in the reorganization of the cytoskeleton, cytokinesis and migration during PDLC differentiation. Conclusions A database of differently displayed proteins during hPDLC osteogenic differentiation was established, which may be helpful to understand and further study molecular mechanism of PDLC osteogenic differentiation.