The Ca2+ selective TRPV6 as well as the L-type Ca2+ channel are regulated by the Ca2+ binding protein calmodulin (CaM). Here we investigated the interaction of CaM with rat (r)TRPV6 in response to alterations of intracellular Ca2+, employing Ca2+-imaging and patch-clamp techniques. Additionally, confocal Förster Resonance Energy Transfer (FRET) microscopy on living cells was utilized as key method to visualize in vivo protein-protein interactions essential for CaM regulation of rTRPV6 activity. The effects of over-expressed CaM or its Ca2+-insensitive mutant (CaMMUT) was probed on various rTRPV6 mutants and fragments in an attempt to elucidate the molecular mechanism of Ca2+/CaM-dependent regulation and to pinpoint the physiologically relevant rTRPV6-CaM interaction site. A significant reduction of rTRPV6 activity as well as an increase in current inactivation was observed when CaM was over-expressed in addition to endogenous CaM. The Ca2+-insensitive CaMMUT, however, failed to affect rTRPV6-derived currents. Accordingly, live cell confocal FRET microscopy revealed a robust interaction for CaM but not CaMMUT with rTRPV6 suggesting a strict Ca2+-dependence for their association. Indeed, interaction of rTRPV6 or its C-terminus with CaM increased with raising intracellular Ca2+ levels as observed by dynamic FRET measurements. An rTRPV6_695-727 mutant with the very C-terminal end deleted yielded Ca2+ currents with a markedly reduced inactivation in accordance with a lack of CaM interaction as substantiated by FRET microscopy. These results in contrast to those for CaM-dependent L-type Ca2+ channel inactivation demonstrate a dynamic association of CaM to the very rTRPV6 C-terminal end (aa 695-727) that enables to accelerate the rate of rTRPV6 current inactivation with increasing intracellular CaM concentrations.