Hyperpolarization-activated calcium channels (HACCs) are found in the plasma membrane and tonoplast of many plant cell types, where they have an important role in Ca2+-dependent signalling. The unusual gating properties of HACCs in plants, i.e., activation by membrane hyperpolarization rather than depolarization, dictates that HACCs are normally open in the physiological hyperpolarized resting membrane potential state (the so-called pump or P-state); thus, if not regulated, they would continuously leak Ca2+ into cells. HACCs are permeable to Ca2+, Ba2+, and Mg2+; activated by H2O2 and the plant hormone abscisic acid (ABA); and their activity in guard cells is greatly reduced by increasing amounts of free cytosolic Ca2+ ([Ca2+]Cyt), and hence closes during [Ca2+]Cyt surges. Here, we demonstrate that the presence of the commonly used Mg-ATP inside the guard cell greatly reduces HACC activity, especially at voltages ≤ −200 mV, and that Mg2+ causes this block. Therefore, we firstly conclude that physiological cytosolic Mg2+ levels affect HACC gating and that channel opening requires either high negative voltages (≥ −200 mV) or displacement of Mg2+ away from the immediate vicinity of the channel. Secondly, based on structural comparisons with a Mg2+-sensitive animal inward-rectifying K+ channel, we propose that the likely candidate HACCs described here are cyclic nucleotide gated channels (CNGCs), many of which also contain a conserved diacidic Mg2+ binding motif within their pores. This conclusion is consistent with the electrophysiological data. Finally, we propose that Mg2+, much like in animal cells, is an important component in Ca2+ signalling and homeostasis in plants.