Pathologic dysregulation of extracellular calciummetabolism is difficult to correct. The extracellular Ca++-sensing receptor (CaSR), a G protein-coupled receptor that regulates renal Ca++ handling through changes in paracellular channel permeability in the thick ascending limb, has emerged as an effective pharmacological candidate for managing calcium metabolism. However, manipulation of CaSR at the systemic level causes promiscuous effects in the parathyroid glands, kidneys, and other tissues, and themechanisms bywhich CaSR regulates paracellular transport in the kidney remain unknown.Here, wedescribe aCaSR-NFATc1-microRNAclaudin-14 signaling pathway in the kidney that underlies paracellular Ca++ reabsorption through the tight junction.WithCaSR-specific pharmacological reagents,we showthat the in vivo gene expression of claudin-14 is regulated through a transcriptional mechanism mediated by NFATc1-microRNA and associated chromatin remodeling. Transgenic knockout and overexpression approaches showed that claudin-14 is required for CaSR-regulated renal Ca++ metabolism. Together, our results define an important signaling cascade that, when dysregulated, may mediate Ca++ imbalance through changes in tight junction permeability.