Objective To investigate whether the low-frequency pulsed electromagnetic fields (LF-PEMFs) can enhance the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) induced by bone morphogenetic protein 9 (BMP9). Methods We isolated CD146⁺/STRO-1⁺ cells (namely PDLSC) from periodontal ligament cells of healthy human premolars, transfected the PDLSC with BMP9-overexpressing recombinant adenoviruses (Ad-GFP-BMP9), exposed the cells to the different intensities of PEMF stimulation (15 Hz, 0.6,1.2,1.8,2.4,3.0 mT, 1 h/12 h), and then detected the expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) by qRT-PCR and Western blotting. Results Overexpression of BMP9 significantly promoted the expression of osteogenic markers (Runx2, ALP, OCN and OPN) in PDLSC (P<0.05). After the intervention with 1.2, 1.8, 2.4 and 3.0 mT PEMF, the expression levels of osteogenic markers in PDLSC were significantly higher than those exposed to BMP9 alone (P<0.05), and reached the peak at 2.4 mT (P<0.05), indicating that LF-PEMFs enhanced BMP9-induced osteogenic differentiation of PDLSC, and there was a "window effect". Conclusion LF-PEMFs stimulation (15 Hz, 1.8 to 3.0 mT) can enhance BMP9-induced osteogenesis of hPDLSCs in vitro.