Cellular mechanisms to account for the low Na⁺ concentration in human milk are poorly defined. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short circuit current (I_sc; a sensitive indicator of net ion transport), suggesting activity of the epithelial Na⁺ channel, ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride sensitive I_sc at all time points tested (2h to 7d), an effect that is not reduced with Ctx washout for 12 hours. Amiloride sensitive Isc remains elevated by Ctx in the presence of inhibitors for PKA (H-89, R_p-cAMP), PI3K (LY294002) and protein trafficking (brefeldin A). Additionally, the Ctx B subunit, alone, does not replicate these effects. RT-PCR and western blot analyses indicate no significant increase in either the mRNA or protein expression for α, β, or, -ENaC subunits. Ctx increases the abundance of both β- and -ENaC in the apical membrane. Additionally, Ctx increases both phosphorylated and nonphosphorylated Nedd4-2 expression. These results demonstrate that human mammary epithelia express ENaC, which can account for the low Na⁺ concentration in milk. Importantly, the results suggest that Ctx increases the expression, but reduces the activity of the E3 ubiquitin ligase, Nedd4-2, which would tend to reduce the ENaC retrieval and increase steady-state membrane residency. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na⁺ transport, which may have inferences for epithelial ion transport regulation in other tissues throughout the body.