Polycystin-2 (PC2) is a Ca2+-regulated Ca2+-channel from the transient receptor potential (TRP) family. Mutations in PC2 can cause autosomal dominant polycystic kidney disease (ADPKD). The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is crucial for channel assembly and function. We have combined biophysical and structural approaches to characterize the Ca2+-dependent molecular mechanism within the C-terminal tail that is involved in channel assembly and functional regulation. We have determined that HPC2 Cterm forms a trimer in solution with and without Ca2+-bound, even though TRP channels are generally tetrameric. We have definitively shown that there is only one Ca2+-binding site in HPC2 Cterm, located within its EF-hand domain. However, its Ca2+-binding affinity is greatly enhanced relative to its intrinsic binding affinity in the isolated EF-hand, possibly due to the positive cooperativity from the trimer interaction. We also employed sea urchin PC2 as a parallel model to study. The sea urchin C-terminal domain construct (SUPC2 Ccore) also trimerizes in solution independent of Ca2+-binding. In contrast, the SUPC2 Ccore contains two Ca2+-binding sites within its EF-hand domain, which exhibit cooperative Ca2+-binding due to internal stabilization. Consequently, trimerization does not further improve Ca2+-binding affinity in SUPC2 Ccore relative to the isolated EF-hand domain. Using both hydrogen-deuterium exchange mass spectroscopy and nuclear magnetic resonance, we have localized the Ca2+-binding sites in PC2 C-terminal tail and mapped the conformational changes induced by Ca2+-binding. We demonstrate that in addition to the direct, local stabilizing effects within the EF-hand, Ca2+-binding also causes conformational changes in the distal coiled-coil domain. This study provides a structural basis for regulation of the PC2 channel by its cytosolic C-terminal domain, with an improved understanding of the functional role of PC2 in regulating intracellular Ca2+ signaling.