Efficient coordination between Mg2+and vitamin D maintains adequate Ca2+levels during lactation. This study explored the possible interaction between Mg2+(0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3(1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and theBGLAPgene product osteocalcin. The mRNA expression ofNT5E,THY1,ENG,SP7,BGLAP,CYP24A1,VDR,SLC41A1,SLC41A2,SLC41A3,TRPM6,TRPM7, andNIPA1was also assessed. Reducing the Mg2+concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression ofCYP24A1was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance ofTHY1,BGLAP, andNIPA1in cells receiving 0.3 mM Mg2+and 5 nM 1,25D. In conclusion, low levels of Mg2+greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+was not modulated by 1,25D, although the expression of certain genes (includingBGLAP) tended to be increased by the combination of low Mg2+and high 1,25D concentrations.