Patent ductus arteriosus (PDA) is the most common cardiovascular abnormality of the preterm infant usually treated with ibuprofen (IBU). PDA is strictly related to oxidative stress (OS) in neonates. This study tests the hypothesis that OS occurs in neonates with PDA and that IBU treatment reduces OS. Forty-three preterm babies with gestational age (GA) <33 weeks were studied prospectively. Three urine samples were collected: at time 0 (before starting treatment), time 1 (after pharmacological PDA closure), and time 2 (7 days after the end of treatment) in all patients. OS was studied by measuring urinary isoprostane (IPs) levels. The results showed significant changes in urinary IP levels from time 0 to time 2 (Kruskal-Wallis, p=0.047). Time trend showed a significant decrease in IPs from time 0 to time 1 after IBU therapy (p=0.0067). This decrease was followed by an increase in IPs levels 7 days after treatment. IBU therapy for PDA closure reduced the risk of OS related to free-radical (FR) generation. This antioxidant effect of IBU may be beneficial in preterm babies with PDA who are at high risk for OS.