Kir4.1 is an inwardly rectifying potassium (K⁺) channel and is expressed in the brain, inner ear, and kidney. In the kidney, Kir4.1 is expressed in the basolateral membrane of the late thick ascending limb (TAL), the distal convoluted tubule (DCT), and the connecting tubule (CNT)/cortical collecting duct (CCD). It plays a role in K⁺ recycling across the basolateral membrane in corresponding nephron segments and in generating negative membrane potential. The renal phenotypes of the loss-function mutations of Kir4.1 include mild salt wasting, hypomagnesemia, hypokalemia, and metabolic alkalosis, suggesting that the disruption of Kir4.1 mainly impairs the transport in the DCT. Patch-clamp experiments and immunostaining demonstrate that Kir4.1 plays a predominant role in determining the basolateral K⁺ conductance in the DCT. However, the function of Kir4.1 in the TAL and CNT/CCD is not essential, because K⁺ channels other than Kir4.1 are also expressed. The downregulation of Kir4.1 in the DCT reduced basolateral chloride (Cl⁻) conductance, suppressed the expression of ste20 proline-alanine-rich kinase (SPAK), and decreased Na-Cl cotransporter (NCC) expression and activity. This suggests that Kir4.1 regulates NCC expression by the modulation of the Cl⁻-sensitive with-no-lysine kinase–SPAK pathway.