microRNAs (miRs) are short (~22 nucleotide) lengths of non-coding RNA that have been implicated in the regulation of post-transcriptional gene expression. Mature miRs incorporate into an inhibitory complex, and bind to recognition sequences on the 3`-untranslated region (UTR) of target mRNA, to prevent protein translation or degrade target mRNA. An upregulation of miR expression is therefore associated with a decrease in target protein levels. The role of miRs in the physiological regulation of sodium transport, and the potential for aldosterone to regulate miR expression has yet to be fully appreciated. There are no reports of miR regulation of the epithelial sodium channel (ENaC). To determine the impact of aldosterone on miR expression in principal cells of the cortical collecting duct (CCD), a microarray analysis was carried out on small RNAs collected from mouse CCD cells treated with 50nM aldosterone for 24 hours. In comparison to non-aldosterone treated controls, the expression levels of 21 miRs were significantly altered (up and down) by aldosterone (n=5). We investigated the three most significantly downregulated miRs, namely mmu-miR-290-5p (0.85 +/- 0.04 fold), mmu-miR-335-3p (0.79 +/- 0.06 fold) and mmu-miR-1983 (0.64 +/- 0.04 fold), as miR downregulation would result in the upregulation of target proteins. The timecourse of miR regulation by aldosterone was monitored by qPCR for these miRs and a reduction in miR expression was observed by 6 hours with maximal inhibition at 12hrs post aldosterone stimulation (n=4). By artificially reducing the expression of these three miRs using transfected antisense locked nucleic acids (LNAs) we were able to increase amiloride-sensitive sodium transport in the mCCD cells by 70.2 +/-4.4% (n=52) without aldosterone stimulation. Overexpression of the three miRs blunted an aldosterone response suggesting that at least part of the aldosterone stimulation of sodium transport occurred through miR signaling. To determine which proteins could be altered by these miRs to regulate sodium transport, in silico predicted protein targets were screened in mCCD cells to test for their involvement in ENaC regulation. One of the candidate miR targets was ankyrin 3 (ank3). Ank3 expression increased in mCCD cells stimulated with 50nM aldosterone for 24hrs. Direct inhibition of miR by LNAs increased ank3 protein levels without aldosterone stimulation indicating that Ank3 was a miR target. To demonstrate the regulation of Ank3 by miRs we transiently transfected the 3′-UTR of ank3 linked to a dual luciferase reporter into mCCD cells. Both aldosterone and changes in miRs themselves altered luciferase expression confirming the UTR as a miR target. Knockdown of ank3 reduced ENaC short-circuit currents compared to control transfected cells (53.9 +/- 5.13%, n=22) and overexpression of ank3 raised amiloride-sensitive short-circuit currents (140.6 +/- 9.75%, n=12), independent of aldosterone or miR regulation. These studies directly linked expression of Ank3 to sodium transport. Ank3 therefore represents a novel aldosterone-induced and miR-regulated protein involved in regulating sodium reabsorption across the CCD.