Voltage-gated calcium (CaV) channels catalyze rapid, highly selective influx of Ca2+ into cells despite a 70-fold higher extracellular concentration of Na+. How CaV channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the tetrameric bacterial NaV channel NaVAb. Our results reveal Ca2+ interactions with two high-affinity Ca2+ binding sites followed by a third lower affinity site that Ca2+ would occupy as it moves inward through the pore. At the entry to the selectivity filter, Site 1 is coordinated by a quartet of the acidic residue, D178, which plays a critical role in determining Ca2+ selectivity. In the center of the selectivity filter, Site 2 is constructed of the carboxyl side chains of D177 and the backbone carbonyls of L176, and is targeted by the blocking cations, Cd2+ and Mn2+, with single occupancy. At the intracellular side of the filter, the backbone carbonyls of T175 form the third, lower affinity site for Ca2+, which mediates exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca2+ 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 CaV channel model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate CaV channels.