High levels of PGE2 have been implicated in the pathogenesis of intestinal inflammatory disorders such as necrotizing enterocolitis (NEC) and peritonitis. However, PGE2 has a paradoxical effect: its low levels promote intestinal homeostasis, whereas high levels may contribute to pathology. These concentration-dependent effects are mediated by four receptors, EP1-EP4. In this study, we evaluate the effect of blockade of the low affinity pro-inflammatory receptors EP1 and EP2 on expression of COX-2, the rate-limiting enzyme in PGE2 biosynthesis, and on gut barrier permeability using cultured enterocytes and three different models of intestinal injury. PGE2 upregulated COX-2 in IEC-6 enterocytes, and this response was blocked by the EP2 antagonist PF-04418948, but not by the EP1 antagonist ONO-8711 or EP4 antagonist E7046. In the neonatal rat model of NEC, EP2 antagonist and low dose of COX-2 inhibitor Celecoxib, but not EP1 antagonist, reduced NEC pathology as well as COX-2 mRNA and protein expression. In the adult mouse endotoxemia and cecal ligation/puncture models, EP2, but not EP1 genetic deficiency decreased COX-2 expression in the intestine. Our results indicate that the EP2 receptor plays a critical role in the positive feedback regulation of intestinal COX-2 by its end-product PGE2 during inflammation and may be a novel therapeutic target in the treatment of NEC.