Objective: To investigate the level changes of miR-150-5p during osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and the effects and possible mechanism of miR-150-5p on hPDLSCs osteogenic differentiation. Methods: hPDLSCs were isolated and cultured by limiting dilution method. Osteogenic induction was performed for hPDLSCs. hPDLSCs were transfected with miR-150-5p-inhibitor and its negative control (miR-inhibitor-NC); the RNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteopontin (OPN), and miR-150-5p were detected by real-time RT-PCR. The protein expression of ALP, Runx2, OPN, β-catenin, TCF-4 and MMP2 was determined by Western blotting. The activity of ALP was measured by alkaline phosphatase detection kit. Results: During osteogenesis induction process, the transcriptional levels of ALP, Runx2, and OPN were gradually increased in hPDLSCs with inductive days (P<0.05), while miR-150-5p level was gradually decreased with inductive days (P<0.05), suggesting an opposite change trend between miR-150-5p level and hPDLSCs osteogenic differentiation. In miR-150-5p-inhibitor transfected hPDLSCs, we observed significant increases of the mRNA and protein levels of ALP, Runx2 and OPN, the protein levels of β-catenin, TCF-4 and MMP2, as well as ALP activity (P<0.05). When hPDLSCs was transfected with miR-150-5p-inhibitor and jointly treated with Wnt signaling pathway inhibitor IWR-1, miR-150-5p downregulation induced increases of ALP, Runx2, OPN and MMP2 expression, and ALP activity were significantly reversed (P<0.05). Conclusion: MiR-150-5p downregulation can facilitate osteogenic differentiation of hPDLSCs and its MMP2 expression via activating Wnt signaling pathway, hinting that the low level of miR-150-5p is beneficial for osteogenic differentiation of hPDLSCs.