Fluid shear stress (FSS) is a critical regulator of cation transport in the collecting duct (CD). High-dietary sodium (Na) consumption increases urine flow, Na excretion, and prostaglandin E₂ (PGE₂) excretion. We hypothesize that increases in FSS elicited by increasing tubular flow rate induce the release of PGE₂ from renal epithelial cells into the extracellular compartment and regulate ion transport. Media retrieved from CD cells exposed to physiologic levels of FSS reveal several fold higher concentration of PGE₂ compared with static controls. Treatment of CD cells with either cyclooxygenase-1 (COX-1) or COX-2 inhibitors during exposure to FSS limited the increase in PGE₂ concentration to an equal extent, suggesting COX-1 and COX-2 contribute equally to FSS-induced PGE₂ release. Cytosolic phospholipase A2 (cPLA2), the principal enzyme that generates the COX substrate arachidonic acid, is regulated by mitogen-activated protein-kinase-dependent phosphorylation and intracellular Ca²⁺ concentration ([Ca²⁺]_i), both signaling processes, of which, are activated by FSS. Inhibition of the ERK and p38 pathways reduced PGE₂ release by 53.3 ± 8.4 and 32.6 ± 11.3%, respectively, while antagonizing the JNK pathway had no effect. In addition, chelation of [Ca²⁺]_i limited the FSS-mediated increase in PGE₂ concentration by 47.5 ± 7.5% of that observed in untreated sheared cells. Sheared cells expressed greater phospho-cPLA2 protein abundance than static cells; however, COX-2 protein expression was unaffected (P = 0.064) by FSS. In microperfused CDs, COX inhibition enhanced flow-stimulated Na reabsorption and abolished flow-stimulated potassium (K) secretion, but did not affect ion transport at a slow flow rate, implicating that high tubular flow activates autocrine/paracrine PGE₂ release and, in turn, regulates flow-stimulated cation transport. In conclusion, FSS activates cPLA2 to generate PGE₂ that regulates flow-mediated Na and K transport in the native CD. We speculate that dietary sodium intake modulates tubular flow rate to regulate paracrine PGE₂ release and cation transport in the CD.