The kidney is the principal organ responsible for the regulation of the body Mg2+ balance. Identification of the gene defect in hypomagnesemia with secondary hypocalcemia recently elucidated transient receptor potential melastatin 6 (TRPM6) as the gatekeeper in transepithelial Mg2+ transport, whereas its homolog, TRPM7, is implicated in cellular Mg2+ homeostasis. The aim of this study was to determine the tissue distribution in mouse and regulation of TRPM6 and TRPM7 by dietary Mg2+ and hormones. This study demonstrates that TRPM6 is expressed predominantly in kidney, lung, cecum, and colon, whereas TRPM7 is distributed ubiquitously. Dietary Mg2+ restriction in mice resulted in hypomagnesemia and renal Mg2+ and Ca2+ conservation, whereas a Mg2+-enriched diet led to increased urinary Mg2+ and Ca2+ excretion. Conversely, Mg2+ restriction significantly upregulated renal TRPM6 mRNA levels, whereas a Mg2+ enriched diet increased TRPM6 mRNA expression in colon. Dietary Mg2+ did not alter TRPM7 mRNA expression in mouse kidney and colon. In addition, it was demonstrated that 17β-estradiol but not 1,25-dihydroxyvitamin D3 or parathyroid hormone regulates TRPM6 renal mRNA levels. Renal TRPM7 mRNA abundance remained unaltered under these conditions. The renal TRPM6 mRNA level in ovariectomized rats was significantly reduced, whereas 17β-estradiol treatment normalized TRPM6 mRNA levels. In conclusion, kidney, lung, cecum, and colon likely constitute the main sites of active Mg2+ (re)absorption in the mouse. In addition, Mg2+ restriction and 17β-estradiol upregulated renal TRPM6 mRNA levels, whereas a Mg2+-enriched diet stimulated TRPM6 mRNA expression in colon, supporting the gatekeeper function of TRPM6 in transepithelial Mg2+ transport.