Prostaglandin E₂ (PGE₂) is the most abundant prostanoid in the kidney, affecting a wide range of renal functions. Conflicting data have been reported regarding the effects of PGE₂ on tubular water and ion transport. The amiloride-sensitive epithelial sodium channel (ENaC) is rate limiting for transepithelial sodium transport in the aldosterone-sensitive distal nephron. The aim of the present study was to explore a potential role of PGE₂ in regulating ENaC in cortical collecting duct (CCD) cells. Short-circuit current (I_SC) measurements were performed using the murine mCCD_cl1 cell line known to express characteristic properties of CCD principal cells and to be responsive to physiological concentrations of aldosterone and vasopressin. PGE₂ stimulated amiloride-sensitive I_SC via basolateral prostaglandin E receptors type 4 (EP₄) with an EC₅₀ of ∼7.1 nM. The rapid stimulatory effect of PGE₂ on I_SC resembled that of vasopressin. A maximum response was reached within minutes, coinciding with an increased abundance of β-ENaC at the apical plasma membrane and elevated cytosolic cAMP levels. The effects of PGE₂ and vasopressin were nonadditive, indicating similar signaling cascades. Exposing mCCD_cl1 cells to aldosterone caused a much slower (∼2 h) increase of the amiloride-sensitive I_SC. Interestingly, the rapid effect of PGE₂ was preserved even after aldosterone stimulation. Furthermore, application of arachidonic acid also increased the amiloride-sensitive I_SC involving basolateral EP₄ receptors. Exposure to arachidonic acid resulted in elevated PGE₂ in the basolateral medium in a cyclooxygenase 1 (COX-1)–dependent manner. These data suggest that in the cortical collecting duct, locally produced and secreted PGE₂ can stimulate ENaC-mediated transepithelial sodium transport.