Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly of prematurely born infants, characterized in its severest from by extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. Proinflammatory cytokines have been implicated in the development of NEC, and we have previously shown that IL-18 is significantly elevated in the well-established neonatal rat model of NEC. To determine whether IL-18 contributes to intestinal pathology in NEC, we subjected IL-18 knockout mice to the protocol used to develop experimental NEC in newborn rats. Newborn B6.129P2- Il18tm1Aki/J (NEC IL-18−/−) and wild-type (NEC WT) mice were hand fed every 3 h with cow's milk-based formula and exposed to asphyxia and cold stress twice daily. After 72 h, animals were killed and distal ileum and liver were removed. Disease development was determined via histological changes in the ileum as scored by a blinded evaluator. The number of TNF-α-, IL-12-, and IL-1β-positive cells and macrophages were determined in both ileum and liver via immunohistology. IκB-α and IκB-β were determined from protein extracts from both ileum and liver using Western blot analysis. The incidence and severity of NEC was significantly reduced in NEC IL-18−/−mice compared with NEC WT. Furthermore, mean ileal macrophages and hepatic IL-1β were significantly reduced in IL-18−/−mice subjected to the NEC protocol. There were no statistically significant changes in Kupffer cells, hepatic TNF-α, ileal IL-1β, or IL-12. IκB-α and IκB-β were significantly increased in NEC IL-18−/−mice ileum and liver, respectively. These results confirm that IL-18 plays a crucial role in experimental NEC pathogenesis.