Klotho, a cofactor in suppressing 1,25(OH) 2 D 3 formation, is a powerful regulator of mineral metabolism. Klotho-hypomorphic mice (kl/kl) exhibit excessive plasma 1,25(OH) 2 D 3 , Ca 2+ , and phosphate concentrations, severe tissue calcification, volume depletion with hyperaldosteronism, and early death. Calcification is paralleled by overexpression of osteoinductive transcription factor Runx2/Cbfa1, Alpl, and senescenceassociated molecules Tgfb1, Pai-1, p21, and Glb1. Here, we show that NH 4 Cl treatment in drinking water (0.28 M) prevented soft tissue and vascular calcification and increased the life span of kl/kl mice .12-fold in males and .4-fold in females without significantly affecting extracellular pH or plasma concentrations of 1,25(OH) 2 D 3 , Ca 2+ , and phosphate. NH 4 Cl treatment significantly decreased plasma aldosterone and antidiuretic hormone concentrations and reversed the increase of Runx2/Cbfa1, Alpl, Tgfb1, Pai-1, p21, and Glb1 expression in aorta of kl/kl mice. Similarly, in primary human aortic smooth muscle cells (HAoSMCs), NH 4 Cl treatment reduced phosphate-induced mRNA expression of RUNX2/CBFA1, ALPL, and senescence-associated molecules. In both kl/kl mice and phosphate-treated HAoSMCs, levels of osmosensitive transcription factor NFAT5 and NFAT5-downstream mediator SOX9 were higher than in controls and decreased after NH 4 Cl treatment. Overexpression of NFAT5 in HAoSMCs mimicked the effect of phosphate and abrogated the effect of NH 4 Cl on SOX9, RUNX2/CBFA1, and ALPL mRNA expression. TGFB1 treatment of HAoSMCs upregulated NFAT5 expression and prevented the decrease of phosphate-induced NFAT5 expression after NH 4 Cl treatment. In conclusion, NH 4 Cl treatment prevents tissue calcification, reduces vascular senescence, and extends survival of klotho-hypomorphic mice. The effects of NH 4 Cl on vascular osteoinduction involve decrease of TGFB1 and inhibition of NFAT5-dependent osteochondrogenic signaling.