PGE, a potent vasodilator, plays a primary role in maintaining the patency of the ductus arteriosus (DA). Genetic disruption of the PGE-specific receptor EP4, however, paradoxically results in fatal patent DA (PDA) in mice. Here we demonstrate that EP4-mediated signals promote DA closure by hyaluronic acid-mediated (HA-mediated) intimal cushion formation (ICF). Chronic EP4 stimulation by ONO-AE1-329, a selective EP4 agonist, significantly enhanced migration and HA production in rat DA smooth muscle cells. When HA production was inhibited, EP4-mediated migration was negated. Activation of EP4, adenylyl cyclase, and PKA all increased HA production and the level of HA synthase 2 (HAS2) transcripts. In immature rat DA explants, ICF was promoted by EP4/PKA stimuli. Furthermore, adenovirus-mediated Has2 gene transfer was sufficient to induce ICF in EP4-disrupted DA explants in which the intimal cushion had not formed. Accordingly, signals through EP4 have 2 essential roles in DA development, namely, vascular dilation and ICF. The latter would lead to luminal narrowing, helping adhesive occlusion and permanent closure of the vascular lumen. Our results imply that HA induction serves as an alternative therapeutic strategy for the treatment of PDA to the current one, i.e., inhibition of PGE signaling by cyclooxygenase inhibitors, which might delay PGE-mediated ICF in immature infants.