Pulsed electromagnetic fields (PEMFs) have been used to treat bone diseases, particularly nonunion healing. Although it is known that PEMFs promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs), to date PEMF molecular mechanisms remain not clearly elucidated. The Notch signalling is a highly conserved pathway that regulates cell fate decisions and skeletal development. The aim of this study was to investigate if the known PEMF-induced osteogenic effects may involve the modulation of the Notch pathway. To this purpose, during in vitro osteogenic differentiation of bone marrow hMSCs in the absence and in the presence of PEMFs, osteogenic markers (alkaline phosphatase activity, osteocalcin and matrix mineralization), the messenger ribonucleic acid expression of osteogenic transcription factors (Runx2, Dlx5, Osterix) as well as of Notch receptors (Notch1-4), their ligands (Jagged1, Dll1 and Dll4) and nuclear target genes (Hes1, Hes5, Hey1, Hey2) were investigated. PEMFs stimulated all osteogenic markers and increased the expression of Notch4, Dll4, Hey1, Hes1 and Hes5 in osteogenic medium compared to control. In the presence of DAPT and SAHM1, used as Notch pathway inhibitors, the expression of the osteogenic markers, including Runx2, Dlx5, Osterix, as well as Hes1 and Hes5 were significantly inhibited, both in unexposed and PEMF-exposed hMSCs. These results suggest that activation of Notch pathway is required for PEMFs-stimulated osteogenic differentiation. These new findings may be useful to improve autologous cell-based regeneration of bone defects in orthopaedics