Inactivation of the thiazide-sensitive sodium chloride cotransporter (NCC) due to genetic mutations in Gitelman's syndrome (GS) or pharmacological inhibition with thiazide diuretics causes hypocalciuria and increased bone mineral density (BMD) with unclear extrarenal calcium (Ca 2þ ) regulation. We investigated intestinal Ca 2þ absorption and bone Ca 2þ metabolism in nonsense Ncc Ser707X (S707X) homozygous knockin mice (Ncc S707X/S707X mice). Compared to wild-type and heterozygous knockin littermates, Ncc S707X/S707X mice had increased intestinal absorption of 45 Ca 2þ and expression of the active Ca 2þ transport machinery (transient receptor potential vanilloid 6, calbindin-D 9K , and plasma membrane Ca 2þ ATPase isoform 1b). Ncc S707X/S707X mice had also significantly increased Ca 2þ content accompanied by greater mineral apposition rate (MAR) in their femurs and higher trabecular bone volume, cortical bone thickness, and BMD determined by mCT. Their osteoblast differentiation markers, such as bone alkaline phosphatase, procollagen I, osteocalcin, and osterix, were also significantly increased while osteoclast activity was unaffected. Analysis of marrowderived bone cells, either treated with thiazide or directly cultured from Ncc S707X knockin mice, showed that the differentiation of osteoblasts was associated with increased phosphorylation of mechanical stress-induced focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). In conclusion, NCC inhibition stimulates duodenal Ca 2þ absorption as well as osteoblast differentiation and bone Ca 2þ storage, possibly through a FAK/ERK dependent mechanism.