Calcium is the most abundant metal in the human body that plays vital roles as a cellular electrolyte as well as the smallest and most frequently used signaling molecule. Calcium uptake in epithelial tissues is mediated by tetrameric calcium-selective transient receptor potential (TRP) channels TRPV6 that are implicated in a variety of human diseases, including numerous forms of cancer. We used TRPV6 crystal structures as templates for molecular dynamics simulations to identify ion binding sites and to study the permeation mechanism of calcium and other ions through TRPV6 channels. We found that at low Ca2+ concentrations, a single calcium ion binds at the selectivity filter narrow constriction formed by aspartates D541 and allows Na+ permeation. In the presence of ions, no water binds to or crosses the pore constriction. At high Ca2+ concentrations, calcium permeates the pore according to the knock-off mechanism that includes formation of a short-lived transition state with three calcium ions bound near D541. For Ba2+, the transition state lives longer and the knock-off permeation occurs slower. Gd3+ binds at D541 tightly, blocks the channel and prevents Na+ from permeating the pore. Our results provide structural foundations for understanding permeation and block in tetrameric calcium-selective ion channels.