Abstract Biodegradable magnesium (Mg) alloys have been investigated for craniofacial and orthopedic bone fracture fixation due to their initial mechanical strength and high biocompatibility. Although Mg alloys have been reported to enhance bone regeneration in vivo, and enhanced osteogenic marker expression in human bone marrow stromal cells (hBMSCs) cultured in Mg alloy extract was reported, however, the biological mechanism is not fully understood. Thus, it is important to elucidate which signaling pathway in the hBMSCs are activated by Mg(2+) that enhances bone formation. We investigated possible mechanisms underlying effects of Mg(2+) on bone regeneration by culturing differentiated and undifferentiated hBMSCs in the presence of culture medium containing 10 mM MgSO4 both with or without osteogenic factors. mRNA expression of osteogenic genes was analyzed using quantitative PCR arrays. Quantitative PCR array data indicated increased mRNA expression of collagen type X and insulin-like growth factor 2, and decreased expression of integrin alpha 3 in the presence of 10 mM MgSO4. Moreover, Western blotting analysis showed enhanced expression of collagen type X, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-2α, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the presence of 10 mM MgSO4. In conclusion, 10 mM of MgSO4 enhanced the production of collagen type X and VEGF by hBMSCs. These results also suggest that Mg(2+) released from bone fixation devices may promote bone regeneration by enhancing the production of collagen type X and VEGF of osteogenic cells in bone tissue.