The transient receptor potential vanilloid channel subfamily member 5 (TRPV5) is a highly selective calcium ion channel predominately expressed in the kidney epithelium that plays an essential role in calcium reabsorption from renal infiltrate. In order to maintain Ca2+ homeostasis, TRPV5 possesses a tightly regulated negative feedback mechanism, where the ubiquitous Ca2+ binding protein calmodulin (CaM) directly binds to the intracellular TRPV5 C-terminus, thus regulating TRPV5. Here we report on the characterization of the TRPV5 C-terminal CaM binding site and its interaction with CaM at an atomistic level. We have solved the de novo solution structure of the TRPV5 C-terminus in complex with a CaM mutant, creating conditions that mimic the cellular basal Ca2+ state. We demonstrate that under these conditions the TRPV5 C-terminus is exclusively bound to the CaM C-lobe only, while it confers conformational freedom to the CaM N-lobe. We also show that at elevated calcium levels, additional interactions between the TRPV5 C-terminus and CaM N-lobe occur, resulting in formation of a tight 1:1 complex, effectively making the N-lobe the calcium sensor. Together, these data are consistent with and support the novel model for Ca2+/CaM-dependent inactivation of TRPV channels as proposed by Bate and co-workers [ Bate , N. , et al. ( 2018 ) Biochemistry , ( 57), DOI: 10.1021/acs.biochem.7b01286 ].