Tight junctions play a key selectivity role in the paracellular conductance of ions. Paracellin-1 is a member of the tight junction claudin protein family and mutations in the paracellin-1 gene cause a human hereditary disease, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) with severe renal Mg²⁺ wasting. The mechanism of paracellin-1 function and its role in FHHNC are not known. Here, we report that in LLC-PK1 epithelial cells paracellin-1 modulated the ion selectivity of the tight junction by selectively and significantly increasing the permeability of Na⁺ (with no effects on Cl^-) and generated a high permeability ratio of Na⁺ to Cl^-. Mutagenesis studies identified a locus of amino acids in paracellin-1 critical for this function. Mg²⁺ flux across cell monolayers showed a far less-pronounced change (compared to monovalent alkali cations) following exogenous protein expression, suggesting that paracellin-1 did not form Mg²⁺-selective paracellular channels. We hypothesize that in the thick ascending limb of the nephron, paracellin-1 dysfunction, with a concomitant loss of cation selectivity, could contribute to the dissipation of the lumen-positive potential that is the driving force for the reabsorption of Mg²⁺.