We have tested the effects of decavanadate (DV), a compound known to interfere with ATP binding in ATP-dependent transport proteins, on TRPM4, a Ca2+-activated, voltage-dependent monovalent cation channel, whose activity is potently blocked by intracellular ATP4−. Application of micromolar Ca2+ concentrations to the cytoplasmic side of inside-out patches led to immediate current activation followed by rapid current decay, which can be explained by an at least 30-fold decreased apparent affinity for Ca2+. Subsequent application of DV (10 μm) strongly affected the voltage-dependent gating of the channel, resulting in large sustained currents over the voltage range between −180 and +140 mV. The effect of DV was half-maximal at a concentration of 1.9 μm. The Ca2+- and voltage-dependent gating of the channel was well described by a sequential kinetic scheme in which Ca2+ binding precedes voltage-dependent gating. The effects of DV could be explained by an action on the voltage-dependent closing step. Surprisingly, DV did not antagonize the effect of ATP4− on TRPM4, but caused a nearly 10-fold increase in the sensitivity of the ATP4− block. TRPM5, which is the most homologous channel to TRPM4, was not modulated by DV. The effect of DV was lost in a TRPM4 chimera in which the C-terminus was substituted with that of TRPM5. Deletion of a cluster in the C-terminus of TRPM4 containing positively charged amino acid residues with a high homology to part of the decavanadate binding site in SERCA pumps, completely abolished the DV effect but also accelerated desensitization. Deletion of a similar site in the N-terminus had no effects on DV responses. These results indicate that the C-terminus of TRPM4 is critically involved in mediating the DV effects. In conclusion, decavanadate modulates TRPM4, but not TRPM5, by inhibiting voltage-dependent closure of the channel.