Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a human disorder caused by mutations in the tight junction protein claudin-16. However, the molecular mechanisms underlining the renal handling of magnesium and its dysfunction causing FHHNC are unknown. Here we show that claudin-16 plays a key role in maintaining the paracellular cation selectivity of the thick ascending limbs of the nephron. Using RNA interference, we have generated claudin-16-deficient mouse models. Claudin-16 knock-down (KD) mice exhibit chronic renal wasting of magnesium and calcium and develop renal nephrocalcinosis. Our data suggest that claudin-16 forms a non-selective paracellular cation channel, rather than a selective Mg(2+)/Ca(2+) channel as previously proposed. Our study highlights the pivotal importance of the tight junction in renal control of ion homeostasis and provides answer to the pathogenesis of FHHNC. We anticipate our study to be a starting point for more sophisticated in vivo analysis of tight junction proteins in renal functions. Furthermore, tight junction proteins could be major targets of drug development for electrolyte disorders.