Magnesium ion (Mg²⁺) is an essential divalent cation and cellular Mg²⁺ concentration is tightly regulated by various Mg²⁺ channels/transporters. Therefore, dysfunction of Mg²⁺ channels/transporters may lead to a variety of cardiovascular or neuromuscular disorders. TRPM7 is a non-selective cation channel, which predominantly permeates Mg²⁺ under physiological conditions. We generated tissue-specific transgenic mouse models expressing the dominant negative TRPM7 mutant (TRPM7DN-Tg) to study the physiological and pathophysiological mechanisms of Mg²⁺ regulation. Whole-cell patch-clamp recordings revealed that TRPM6/7 currents in HEK293 cells were almost completely attenuated by co-expression of TRPM7DN mutant. Renal tubule-specific TRPM7DN-Tg exhibited dysregulation of serum Mg²⁺ level and urinary Mg²⁺ excretion. Interestingly, in these mice, phenylephrine (PE)-induced vascular contractile responses was significantly attenuated. On the other hand, vascular smooth muscle-specific TRPM7DN-Tg showed attenuation of PE-induced contractile responses without changing serum Mg²⁺ level and urinary Mg²⁺ excretion. These results suggest that TRPM6/7 channels are tissue-dependently involved in the regulation of Mg²⁺ homeostasis and vascular contraction. Our tissue-specific TRPM7DN-Tg will be useful animal models for studying magnesium disorders.