Mammary epithelia produce an isotonic, low Na⁺ fluid that is rich in nutrients. Mechanisms that account for the low electrolyte concentration have not been elucidated, although amiloride-sensitive ion transport has been reported in some situations. We hypothesized that corticosteroid exposure modulates epithelial Na⁺ channel (ENaC) expression and/or activity in bovine mammary epithelial cells. BME-UV cells were grown to confluent monolayers on permeable supports with a standard basolateral medium and apical medium of low electrolyte-high lactose composition that resembles the ionic composition of milk. Ion transport was assessed in modified Ussing flux chambers. Exposure to glucocorticoids (dexamethasone, cortisol, or prednisolone), but not aldosterone, increased short circuit current (I_sc), a sensitive measure of net ion transport, while apical exposure to amiloride or benzamil reduced corticosteroid-induced I_sc close to basal levels. Quantitative RT-PCR indicated a glucocorticoid-induced increase in mRNA for the - and - subunits of ENaC, whereas -ENaC mRNA expression was only mildly affected. Exposure to mifepristone (a glucocorticoid receptor antagonist), but not spironolactone (a mineralocorticoid receptor antagonist), precluded both the corticosteroid-induced elevation in amiloride-sensitive I_sc and the induced changes in - and -ENaC mRNA. We conclude that Na⁺ movement across mammary epithelia is modulated by corticosteroids via a glucocorticoid receptor-mediated mechanism that regulates expression of the - and -ENaC subunits. ENaC expression and activity could account for the low Na⁺ concentration that is typical of milk.