Transient receptor potential cation channel subfamily M member 7 (TRPM7) is an ion channel/protein kinase belonging to the TRP melastatin and eEF2 kinase families. Under physiological conditions, most native TRPM7 channels are inhibited by cytoplasmic Mg2+, protons, and polyamines. Currents through these channels (ITRPM7) are robustly potentiated when the cell interior is exchanged with low Mg2+-containing buffers. ITRPM7 is also potentiated by phosphatidyl inositol bisphosphate (PI(4,5)P2) and suppressed by its hydrolysis. Here we characterized internal Mg2+- and pH-mediated inhibition of TRPM7 channels in HEK293 cells overexpressing WT voltage-sensing phospholipid phosphatase (VSP) or its catalytically inactive variant VSP-C363S. VSP-mediated depletion of membrane phosphoinositides significantly increased channel sensitivity to Mg2+ and pH. Proton concentrations that were too low to inhibit ITRPM7 when the VSP-C363S variant was expressed (pH 8.2) became inhibitory in WT VSP–expressing cells. At pH 6.5, protons inhibited ITRPM7 both in WT and VSP C363S–expressing cells but with a faster time course in the WT VSP–expressing cells. Inhibition by 150 μM Mg2+ was also significantly faster in the WT VSP–expressing cells. Cellular PI(4,5)P2 depletion increased the sensitivity of TRPM7 channels to the inhibitor 2-aminoethyl diphenyl borinate, which acidifies the cytosol. Single substitutions at Ser-1107 of TRPM7, reducing its sensitivity to Mg2+, also decreased its inhibition by spermine and acidic pH. Furthermore, these channel variants were markedly less sensitive to VSP-mediated PI(4,5)P2 depletion than the WT. We conclude that the internal Mg2+-, polyamine-, and pH-mediated inhibition of TRPM7 channels is not direct but, rather, reflects electrostatic screening and resultant disruption of PI(4,5)P2–channel interactions.