The dysfunction of Na+-Cl− cotransporter (NCC) caused by mutations in solute carrier family12, member 3 gene (SLC12A3) primarily causes Gitelman syndrome (GS). In identifying the pathogenicity of R158Q and G212S variants of SLC12A3, we evaluated the pathogenicity by bioinformatic, expression, and localization analysis of two variants from a patient in our cohort. The prediction of mutant protein showed that p.R158Q and p.G212S could alter protein’s three-dimensional structure. Western blot showed a decrease of mutant Ncc. Immunofluorescence of the two mutations revealed a diffuse positive staining below the plasma membrane. Meanwhile, we conducted a compound heterozygous model—Ncc R156Q/G210S mice corresponding to human NCC R158Q/G212S. NccR156Q/G210S mice clearly exhibited typical GS features, including hypokalemia, hypomagnesemia, and increased fractional excretion of K+ and Mg2+ with a normal blood pressure level, which made NccR156Q/G210S mice an optimal mouse model for further study of GS. A dramatic decrease and abnormal localization of the mutant Ncc in distal convoluted tubules contributed to the phenotype. The hydrochlorothiazide test showed a loss of function of mutant Ncc in NccR156Q/G210S mice. These findings indicated that R158Q and G212S variants of SLC12A3 were pathogenic variants of GS.