Voltage-gated calcium (Ca V) channels catalyze rapid, highly selective influx of Ca 2+ into cells despite 70-fold higher extracellular concentration of Na +. How Ca V channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial Na V channel Na VAb. Our results reveal interactions of hydrated Ca 2+ with two high-affinity Ca 2+-binding sites followed by a third lower-affinity site that would coordinate Ca 2+ as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side-chains, which play a critical role in determining Ca 2+ selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations, Cd 2+ and Mn 2+, with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca 2+ ions bound in the selectivity filter and supports a “knock-off” mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our Ca VAb model establishes a structural framework for understanding mechanisms of ion selectivity and conductance by vertebrate Ca V channels.