Abstract: Magnesium (Mg²⁺) is an essential metal for plant growth; however, its over-accumulation in cells can be cytotoxic. The metal tolerance protein family (MTP) belongs to an ubiquitous family of cation diffusion facilitator (CDF) proteins that export divalent metal cations for metal homeostasis and tolerance in all organisms. We describe here the identification of MTP10 to be critical for xylem Mg homeostasis in Arabidopsis under high Mg²⁺ conditions. The Arabidopsis plant contains 12 MTP genes, and only knockout of MTP10 decreased the tolerance of high-Mg stress. The functional complementation assays in a Mg²⁺ -uptake-deficient bacterial strain MM281 confirmed that MTP10 conducted Mg²⁺ transport. MTP10 is localized to the plasma membrane of parenchyma cells around the xylem. Reciprocal grafting analysis further demonstrated that MTP10 functions in the shoot to determine the shoot growth phenotypes under high Mg²⁺ conditions. Moreover, compared to the wild type, the mtp10 mutant accumulated more Mg²⁺ in xylem sap under high-Mg stress. This study reveals that MTP10 facilitates Mg²⁺ diffusion from the xylem to shoots and thus determines Mg homeostasis in shoot vascular tissues during high-Mg stress.