Objective To investigate the effects of enamel matrix derivative (EMD) on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and the potential mechanism. Methods The primary cultured hPDLSCs were identified by flow cytometry, and the third generation cells were selected for experiments. The cell counting kit-8 assay was performed to detect the effect of different EMD concentrations (0, 20, 50 and 100 mg·L-1) on the proliferation of hPDLSCs. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the effect of EMD on the osteogenic differentiation of hPDLSCs in different concentrations (0, 20, 50 and 100 mg·L-1). The effect of different EMD concentrations (0, 20, 50 and 100 mg·L-1) on collagen synjournal and mineralised nodule formation of hPDLSCs were detected through trichrome and Von Kosa’s staining. Western blotting and qRT-PCR were performed to detect β-catenin, RunX2, CaMKⅡ and NLK expressions after different concentrations of EMD and DDK1 were applied to hPDLSCs.Results EMD significantly promoted the proliferation of hPDLSCs, which increased with the increase in concentration and stimulation time of EMD. After EMD treatment with hPDLSCs, the formation of mineralised nodules and collagen synjournal increased significantly. Furthermore, the expressions of osteocalcin, collagenⅠand RunX2 were elevated. Moreover, the expressions of β-catenin, RunX2, CaMKⅡ and NLK were significantly increased with EMD treatment and inhibited by DDK1.Conclusion EMD can promote the proliferation and osteogenic differentiation of hPDLSCs in vitro through the Wnt/β-catenin signalling pathway.