Aldosterone is a major regulator of epithelial Na⁺ absorption. One of its principal targets is the epithelial Na⁺ channel α-subunit (ENaCα), principally expressed in the kidney collecting duct, lung, and colon. Models of aldosterone-mediated trans-activation of the ENaCα gene have focused primarily on interactions of liganded nuclear receptors with the ENaCα gene promoter. Herein, we demonstrate that the murine histone H3 lysine-79 methyltransferase, murine disruptor of telomeric silencing alternative splice variant “a” (mDot1a), is a novel component in the aldosterone signaling network controlling transcription of the ENaCα gene. Aldosterone downregulated mDot1a mRNA levels in murine inner medullary collecting ducts cells, which was associated with histone H3 K79 hypomethylation in bulk histones and at specific sites in the ENaCα 5′-flanking region, and trans-activation of ENaCα. Knockdown of mDot1a by RNA interference increased activity of a stably integrated ENaCα promoter-luciferase construct and expression of endogenous ENaCα mRNA. Conversely, overexpression of EGFP-tagged mDot1a resulted in hypermethylation of histone H3 K79 at the endogenous ENaCα promoter, repression of endogenous ENaCα mRNA expression, and decreased activity of the ENaCα promoter-luciferase construct. mDot1a-mediated histone H3 K79 hypermethylation and repression of ENaCα promoter activity was abolished by mDot1a mutations that eliminate its methyltransferase activity. Collectively, our data identify mDot1a as a novel aldosterone-regulated histone modification enzyme, and, through binding the ENaCα promoter and hypermethylating histone H3 K79 associated with the ENaCα promoter, a negative regulator of ENaCα transcription.