The mechanisms by which prostanoids contribute to the maintenance of whole body water homeostasis are complex and not fully understood. The present study demonstrates that an EP3-dependent feedback mechanism contributes to the regulation of water homeostasis under high salt conditions. Rats on a normal diet and tap water were placed in metabolic cages and given either sulprostone (20 µg/kg/day) or vehicle for 3 days to activate EP3 receptors in thick ascending limb (TAL). Treatment was continued for another 3 days in rats given either 1% NaCl in the drinking water or tap water. Sulprostone decreased expression of COX-2 expression by approximately 75% in TAL tubules from rats given 1% NaCl concomitant with a ~60% inhibition of COX-2-dependent PGE2 levels in the kidney. Urine volume increased after ingestion of 1% NaCl, but was reduced ~40% by sulprostone. In contrast, the highly selective EP3 receptor antagonist (L-798106, 100 µg/kg/day), which increased COX-2 expression and renal PGE2 production, increased urine volume in rats given 1% NaCl. Sulprostone increased expression of AQP2 in inner medullary collecting duct plasma membrane in association with an increase in phosphorylation at Ser269 and decrease in Ser261 phosphorylation; antagonism of EP3 with L798106 reduced AQP2 expression. Thus, although acute activation of EP3 by PGE2 in the TAL and collecting duct inhibits NKCC2 and AQP2 activity, respectively, chronic activation of EP3 in vivo limits the extent of COX-2-derived PGE2 synthesis thereby mitigating the inhibitory effects of PGE2 on these transporters and decreasing urine volume.