TRPV5 is unique within the large TRP channel family for displaying a high Ca2+ selectivity together with Ca2+-dependent inactivation. Our study aims to uncover novel insights into channel gating through in-depth structure-function analysis. We identify an exceptional tryptophan (W583) at the terminus of the intracellular pore that is unique for TRPV5 (and TRPV6). A combination of site-directed mutagenesis, biochemical and electrophysiological analysis, together with homology modeling, demonstrates that W583 is part of the gate for Ca2+ permeation. The W583 mutants show increased cell death due to profoundly enhanced Ca2+ influx, resulting from altered channel function. A glycine residue above W583 might act as flexible linker to rearrange the tryptophan gate. Furthermore, we hypothesize functional crosstalk between the pore region and carboxy terminus, involved in Ca2+-calmodulin-mediated inactivation. This study proposes a unique channel gating mechanism and delivers detailed molecular insight into the Ca2+ permeation pathway that can be extrapolated to other Ca2+-selective channels.