Aldosterone is a key regulator of Na+ conservation in the kidney through rapid non-genomic and genomic actions on the epithelial sodium channel (ENaC) (1). ENaC activity is the major determinant of the rate of Na+ reabsorption within the renal cortical collecting duct (CCD). Here we report a novel mechanism for aldosterone regulation of ENaC activity and sub-cellular trafficking via protein kinase D2 (PKD2) in a murine cortical collecting duct (M1-CCD) cell line. ENaC current was measured as the amiloride-inhibited transepithelial short-circuit current (ISC) in M1-CCD cell monolayers mounted in Ussing chambers. ENaC and PKD2 sub-cellular localization were assessed by immunofluorescence confocal microscopy. Student’s t-test was performed with one way ANOVA. Aldosterone (10nM) caused a rapid phosphorylation of PKD2 (within 10min) accompanied by a subcellular redistribution of PKD2 from the apical membrane into the cytosol. Activation of PKD2 correlated with an increased abundance of ENaCγ at the apical membrane, an increase in Nedd4-2 phosphorylation and a 3-fold stimulation of the amiloride-sensitive ISC. Suppressing PKD2 expression by shRNA in M1-CCD cells resulted in an increase in ENaC abundance at the apical membrane and an 8-fold stimulation of the ISC. Paradoxically, aldosterone treatment resulted in a 4-fold decrease in ISC in the M1-CCD PKD2 knock-down cells. Our results show that PKD2 is a newly-discovered inhibitory regulator of basal ENaC stability in the apical membrane, possibly by modulating ubiquitination of the channel and this inhibition is removed by aldosterone-induced phosphorylation of PKD2 and sequestration in the cytosol. Knock down PKD2 expression caused aldosterone to be inhibitory of Na+ reabsorption and ENaC abundance in the apical membrane, thus exposing a previously unknown pleiotropic effect of aldosterone on ENaC membrane stability and channel conductance. We propose that protein kinase D isoforms are signalling molecules controlling both the steady-state and aldosterone-induced membrane stability of ENaC and represent a novel important signal transduction mechanism for aldosterone regulation of renal Na+ reabsorption.