Human mesenchymal stem cells (hMSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications by virtue of their capacity for self-renewal and multipotent differentiation. Our intent was to characterize the effect of pulsed electromagnetic fields (PEMFs) on the proliferation and osteogenic differentiation of hMSCs in vitro. hMSCs isolated from the bone marrow of adult patients were cultured with osteogenic medium for up to 28 days and exposed to daily PEMF stimulation with single, narrow 300 micros quasi-rectangular pulses with a repetition rate of 7.5 Hz. Relatively greater cell numbers were observed at late stages of osteogenic culture with PEMF exposure. The production of alkaline phosphatase (ALP), an early marker of osteogenesis, was significantly enhanced at day 7 with PEMF treatment in both basal and osteogenic cultures as compared to untreated controls. Furthermore, the expressions of other early osteogenic genes, including Runx2/Cbfa1 and ALP, were also partially modulated by PEMF exposure, indicating that osteogenesis in hMSCs was associated with the specific PEMF stimulation. Based on ALP and alizarin red S staining, the accumulation of ALP protein produced by the hMSCs as well as calcium deposits reached their highest levels at day 28. Our results indicate that extremely low-frequency PEMF stimulation may play a modulating role in hMSC osteogenesis. Taken together, these findings provide insights on the development of PEMF as an effective technology for regenerative medicine.