Electromagnetic fields (EMFs) are widely used in a number of cell therapies and bone disorder treatments, and nanomagnetic particles (NMPs) also promote cell activity. In this study, we investigated the synergistic effects of EMFs and NMPs on the osteogenesis of the human Saos-2 osteoblast cell line and in a rat calvarial defect model. The Saos-2 cells and critical-size calvarial defects of the rats were exposed to EMF (1 mT, 45 Hz, 8 h/day) with or without Fe O NMPs. Biocompatibility was evaluated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) assays. This analysis showed that NMP and EMF did not induce cell toxicity. Quantitative reverse-transcription polymerase chain reaction indicated that the osteogenesis-related markers were highly expressed in the NMP-incorporated Saos-2 cells after exposure to EMF. Also, the expression of gene-encoding proteins involved in calcium channels was activated and the calcium concentration of the NMP-incorporated + EMF-exposed group was increased compared with the control group. In particular, in the NMP-incorporated + EMF-exposed group, all osteogenic proteins were more abundantly expressed than in the control group. This indicated that the NMP incorporation + EMF exposure induced a signaling pathway through activation of p-ERK and calcium channels. Also, in vivo evaluation revealed that rat calvarial defects treated with EMFs and NMPs had good regeneration results with new bone formation and increased mineral density after 6 weeks. Altogether, these results suggest that NMP treatment or EMF exposure of Saos-2 cells can increase osteogenic activity and NMP incorporation following EMF exposure which is synergistically efficient for osteogenesis.