Magnesium (Mg²⁺) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg²⁺ transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3, as major mechanisms of Mg²⁺ influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg²⁺. Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg²⁺ concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg²⁺ or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg²⁺ transport system.