Aldosterone is a major regulator of epithelial Na⁺ absorption. One of its principal targets is the epithelial Na⁺ channel -subunit (ENaC) principally expressed in kidney collecting duct, lung, and colon. Models of aldosterone-mediated transactivation of ENaC have focused primarily on interactions of liganded nuclear receptors with the ENaC promoter. Here, 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 ENaC gene. Aldosterone down-regulated mDot1a mRNA levels in mIMCD3 cells, which was associated with histone H3 K79 hypomethylation in bulk histones and at specific sites in the ENaC 5'-flanking region, and transactivation of ENaC. Knockdown of mDot1a by RNA interference increased activity of a stably integrated ENaC promoter-luciferase construct and expression of endogenous ENaC mRNA. Conversely, overexpression of enhanced green fluorescent protein (EGFP)-tagged mDot1a resulted in its association with, and hypermethylation of histone H3 K79 at the endogenous ENaC promoter, repression of endogenous ENaC mRNA expression, and decreased activity of the ENaC promoter-luciferase construct. mDot1a-mediated histone H3 K79 hypermethylation and repression of ENaC 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 ENaC promoter and hypermethylating histone H3 K79 associated with the ENaC promoter, a negative regulator of ENaC transcription.