Large-conductance voltage- and Ca²⁺-dependent K⁺ (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca²⁺ homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the “gating ring”), which confers sensitivity to intracellular Ca²⁺ and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca²⁺- and Mg²⁺-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba²⁺-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca²⁺-binding site (the “calcium bowl”) reduced the Ca²⁺ and abolished the Mg²⁺ dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca²⁺-binding site and that Mg²⁺ can bind to the calcium bowl with less affinity than Ca²⁺. Dynamic light scattering analysis revealed a reversible Ca²⁺-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.