The transient receptor potential vanilloid 5 (TRPV5) channel is a highly calcium selective ion channel that regulates systemic calcium homeostasis by acting as a critical gate for calcium reabsorption in the kidney. Human polymorphisms of this channel have exemplified the importance of TRPV5 in disorders of calcium homeostasis, but atomic level information regarding this channel had remained unknown. To address this gap, we utilized cryo-electron microscopy (cryo-EM) coupled with computational and biochemical approaches to understand the gating and modulation of TRPV5. First, we investigated the mechanism of action of the inhibitor econazole on TRPV5 which yielded the first reported structure of this protein. From that structure we were able to identify the econazole binding site and gain insights into how binding of a modulator in that pocket would result in channel inhibition. We then sought to elucidate the mechanism of action of endogenous modulators of TRPV5 including the intracellular calcium binding protein calmodulin (CaM) and the abundant plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) which act as an inhibitor and activator, respectively. By solving the structures of TRPV5 in the presence of these modulators as well as TRPV5 in an apo state we clearly identified the binding regions for these two modulators and the conformational changes that confer inhibition or activation. Finally, using the structural information that we had gathered coupled with structure-based virtual screening we uncovered two novel inhibitors of TRPV5 both of which bound in different sites on the protein and revealed novel mechanisms of inhibition of TRPV5. These studies have thus expanded the base of understanding of TRPV5 at the atomic level and laid the foundation for future studies including but not limited to rational drug design, molecular dynamics investigations and targeted biochemical probing.