Arterial calcification, common in vascular diseases, involves vascular smooth muscle cell (VSMC) transformation to an osteoblast phenotype. Clinical studies suggest that magnesium may prevent this, but mechanisms are unclear. We assessed whether increasing magnesium levels reduce VSMC calcification and differentiation and questioned the role of the Mg(2+) transporter, transient receptor potential melastatin (TRPM)7 cation channels in this process. Rat VSMCs were exposed to calcification medium in the absence and presence of magnesium (2.0 to 3.0 mmol/L) or 2-aminoethoxy-diphenylborate (2-APB) (TRPM7 inhibitor). VSMCs from mice with genetically low (MgL) or high-normal (MgH) [Mg(2+)](i) were also studied. Calcification was assessed by von Kossa staining. Expression of osteocalcin, osteopontin, bone morphogenetic protein (BMP)-2, BMP-4, BMP-7, and matrix Gla protein and activity of TRPM7 (cytosol:membrane translocation) were determined by immunoblotting. Calcification medium induced osteogenic differentiation, reduced matrix Gla protein content, and increased expression of the sodium-dependent cotransporter Pit-1. Magnesium prevented calcification and decreased osteocalcin expression and BMP-2 activity and increased expression of calcification inhibitors, osteopontin and matrix Gla protein. TRPM 7 activation was decreased by calcification medium, an effect reversed by magnesium. 2-APB recapitulated the VSMC osteoblastic phenotype in VSMCs. Osteocalcin was increased by calcification medium in VSMCs and intact vessels from MgL but not MgH, whereas osteopontin was increased in MgH, but not in MgL mice. Magnesium negatively regulates vascular calcification and osteogenic differentiation through increased/restored TRPM7 activity and increased expression of anticalcification proteins, including osteopontin, BMP-7, and matrix Gla protein. New molecular insights are provided whereby magnesium could protect against VSMC calcification.