Background: Despite technical and scientific improvements in neonatal medicine, there is still no efficient treatment for or prevention of neonatal necrotizing enterocolitis (NEC). Objective: The aim of this study was to determine the protective effect of surfactant on intestinal cells in hypoxia-induced experimental NEC in rats. Methods: The study was performed on Sprague Dawley rat pups divided into three groups. Group 1 served as nonhypoxic controls (n = 15). Group 2 rats (untreated; n = 15) were subjected to hypoxia and reoxygenation (H/O). Group 3 rats (surfactant treated; n = 15) were treated with surfactant (Curosurf; 400 mg/kg enterally) for 5 days and then subjected to H/O. All animals were killed on day 5, and histological and biochemical examinations were performed on intestinal samples. Results: There were no histopathological changes in the control group. The histological findings in untreated rats were similar to those seen in neonatal NEC, with destruction of villi and crypts and extension to the muscularis layer. The mean histological grade of group 1 was 0.2 (range 0–1), and in group 2, it was 2.1 (range 1–3). There was no statistical difference between the histological grades of groups 2 and 3 (p > 0.05). However, there was a difference between the groups that did and did not receive surfactant with regard to superoxide dismutase (SOD), glutathione peroxidase (GSH) and malondialdehyde (MDA). Conclusion: Although surfactant did not have a protective effect on histological changes in hypoxic neonatal rat intestine, it improves biochemical markers such as SOD and GSH and reduces MDA levels.