Summary Two‐pore channels (TPC) have been established as components of calcium signalling networks in plants and animals. In plants, TPC1 in the vacuolar membrane is gated open upon binding of calcium in a voltage‐dependent manner. Here, we analyzed the molecular mechanism of the Ca2+‐dependent activity of TPC1 from Arabidopsis thaliana, using site‐directed mutagenesis of its two canonical EF‐hands. Wild‐type TPC1 and TPC1‐D335A with a mutated first Ca2+ ligand in EF‐hand 1 produced channels that retained their voltage‐ and Ca2+‐dependent gating characteristics, but were less sensitive at Ca2+ concentrations <200 μm. Additional mutation of the first Ca2+ ligand in EF‐hand 2 resulted in silent TPC1‐D335A/D376A channels. Similarly, the single mutant TPC1‐D376A could not be activated up to 1 mm Ca2+, indicating that the second EF‐hand is essential for the Ca2+‐dependent channel gating. Molecular modeling suggests that EF‐hand 1 displays a low‐affinity Ca2+/Mg2+‐binding site, while EF‐hand 2 represents a high‐affinity Ca2+‐binding site. Together, our data prove that EF‐hand 2 is responsible for the Ca2+‐receptor characteristics of TPC1, while EF‐hand 1 is a structural site required to enable channel responses at physiological changes in Ca2+ concentration.