Objective To explore whether c-Jun N-terminal kinase (JNK) pathway is involved in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) induced by low-frequency pulsed electromagnetic field (PEMF) via using the specific inhibitor SP600125 to intervene JNK signaling pathway. Methods hPDLSCs were exposed to the low-frequency PEMF stimulation (15 Hz, 0-3.0 mT, radiate for 1 h every 12 h) in vitro for 7 d or 14 d and the expression levels of osteogenic-related genes Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) were detected by quantitative polymerase chain reaction (qPCR), so as to determine the osteogenic differentiation ability of cells induced by low-frequency PEMF and the appropriate magnetic field intensity. To determine whether JNK pathway plays a role in the osteogenic differentiation of hPDLSCs stimulated by low-frequency PEMF, the expression levels of JNK and phosphorylated-JNK (p-JNK) proteins were detected by Western blotting; and the expression levels of osteogenic genes in cells treated with different concentrations of SP600125 were detected by qPCR. Results The expression levels of osteogenic-related genes Runx2, ALP, OPN and OCN were higher in hPDLSCs irradiated with low-frequency PEMF at 15 Hz and 2.5 mT than other intensity groups (all P<0.05). Low-frequency PEMF significantly stimulated the expression of JNK and p-JNK proteins in cells (both P<0.05). The expression levels of osteogenic genes in hPDLSCs were decreased after the JNK pathway was inhibited by SP600125, and the inhibitory effects of 20 and 30 μmol/L SP600125 were more obvious than that of 10 μmol/L SP600125 (both P<0.05). Conclusion PEMF (15 Hz,2.5 mT) partially activates JNK pathway to induce hPDLSC osteogenic differentiation.