Electromagnetic field (EMF) stimulation is clinically beneficial for fracture nonunion and a wide range of bone disorders. However, no consensus has been reached on the optimal parameters of the EMF. The exact mechanism by which EMFs enhance osteogenesis has also not been defined. In the present study, a sinusoidal 1 mT EMF at frequencies of 10, 30, 50, and 70 Hz were administered to rat bone marrow mesenchymal stromal cells (rBMSCs) in the cyclic mode of 2 h exposures followed by 4 h of culture without exposure. The cell viability, proliferation, expression of some osteogenic genes, and mineralization of the extracellular matrix were investigated. It was found that the cell viability was decreased by EMF exposures of 50 and 70 Hz. The proliferation of rBMSCs was elevated significantly in the 10 Hz EMF-treated group during the culture periods. The expression of alkaline phosphatase (ALP) and osteocalcin (OC), two early-phase osteogenic differentiation markers, was up-regulated by the 1 mT, 10 Hz EMF after 1 week. However, the expression of genes that marked the later-phase osteogenic differentiation and maturation of osteoblasts was elevated by the stimulation of 50 Hz EMFs after 2 weeks. In addition, it was observed that the mineralization of the extracellular matrix was enhanced by 50 Hz EMF exposure. These results indicated that the 1 mT EMF at different frequencies had disparate effects on the viability, proliferation and osteogenic differentiation of rBMSCs, and may be beneficial for developing novel therapeutic approaches in bone regenerative medicine. Bioelectromagnetics. 34:453–464. © 2013 Wiley Periodicals, Inc.