Claudin-3 is a tight junction protein expressed in the distal nephron, notably in the thick ascending limb of Henle’s loop and the distal convoluted tubule, where it acts as an aldosterone-regulated paracellular barrier to sodium and chloride transport. Here, we demonstrate that claudin-3 knockout (KO) mice exhibit an unexpected diet-dependent alteration in glomerular filtration rate (GFR): under a normal sodium (NS) diet, GFR decreases significantly, whereas under a low sodium (LS) diet, GFR increases paradoxically compared with wild-type controls. To identify the underlying mechanism, we examined claudin-3 expression in the macula densa , the specialized epithelial structure located at the junction between the thick ascending limb and the distal convoluted tubule that governs tubuloglomerular feedback (TGF). Immunofluorescence analysis revealed that claudin-3 colocalizes with neuronal nitric oxide synthase (nNOS) and the Na ⁺ -K ⁺ -2Cl ^- cotransporter (NKCC2) in a distinct cluster of cells characterized by densely packed nuclei, positioned in direct contact with the glomerulus and immediately adjacent to an NCC-positive distal convoluted tubule, while remaining itself NCC-negative. Notably, within the tubule containing the macula densa , nNOS expression was strictly confined to macula densa cells, clearly distinguishing them from the surrounding tubular epithelium. These results identify claudin-3 as a previously unrecognized component of macula densa biology and suggest a role for claudin-3 in the control of GFR possibly via the tubuloglomerular feedback.