TRPV5 is a Ca2+-selective channel that plays a key role in the reabsorption of Ca2+ ions in the kidney. Recently, a rare L530R variation (rs757494578) of TRPV5 was found to be associated with recurrent kidney stones in a founder population. However, it was unclear to what extent this variation alters the structure and function of TRPV5. To evaluate the function and expression of the TRPV5 variant, Ca2+ uptake in Xenopus oocytes and western blot analysis were performed. The L530R variation abolished the Ca2+ uptake activity of TRPV5 in Xenopus oocytes. The variant protein was expressed with drastic reduction in complex glycosylation. To assess the structural effects of this L530R variation, TRPV5 was modeled based on the crystal structure of TRPV6 and molecular dynamics simulations were carried out. Simulation results showed that the L530R variation disrupts the hydrophobic interaction between L530 and L502, damaging the secondary structure of transmembrane domain 5. The variation also alters its interaction with membrane lipid molecules. Compared to the electroneutral L530, the positively charged R530 residue shifts the surface electrostatic potential towards positive. R530 is attracted to the negatively charged phosphate group rather than the hydrophobic carbon atoms of membrane lipids. This shifts the pore helix where R530 is located and the D542 residue in the Ca2+-selective filter towards the surface of the membrane. These alterations may lead to misfolding of TRPV5, reduction in translocation of the channel to the plasma membrane and/or impaired Ca2+ transport function of the channel, and ultimately disrupt TRPV5-mediated Ca2+ reabsorption.