The TRPV5 gene, which encodes the Ca²⁺ channel in the apical membrane of distal convoluted tubule and connecting tubule of the kidney, exhibits unusually high frequency of nonsynonymous single nucleotide polymorphisms (SNPs) among African Americans. To assess the functional impacts of the nonsynonymous SNP variations in TRPV5, these variants were analyzed with radiotracer ⁴⁵Ca²⁺ influx assay and voltage clamp technique using Xenopus laevis oocytes. Among the variations tested, including A8V, R154H, A563T and L712F, the latter two significantly increased TRPV5-mediated Ca²⁺ influx. The A563T variant, which exists in African Americans with relative high frequency, exhibited increased Ca²⁺ influx at extracellular Ca²⁺ from 0.01 to 2 mM despite a lower expression level at the plasma membrane. This variant also exhibited a reduction in Na⁺ current as a result of increased sensitivity to extracellular Mg²⁺. By substituting threonine 563 (Thr563) with serine or valine residue, the bulky side chain of Thr563 was shown to facilitate Ca²⁺ transport, whereas the hydroxyl group of Thr563 is likely related to Mg²⁺ sensitivity. The A563T variant was capable of increasing TRPV5-mediated Ca²⁺ influx, even when it was expressed under condition mimicking heterozygous or compound state with other variants. In conclusion, the A563T variant of TRPV5 significantly increased Ca²⁺ influx by affecting the Ca²⁺ permeation pathway. Thus, the A563T variation in TRPV5 gene may contribute to the superior ability of renal Ca²⁺ conservation in African Americans.