Background & Aims: High-mobility group box-1 (HMGB1) is a late mediator of endotoxemia known to stimulate the production of pro-inflammatory cytokines that are putative mediators of intestinal inflammation associated with necrotizing enterocolitis (NEC). We hypothesized that HMGB1 is also involved in the pathogenesis of NEC. We examined the expression of HMGB1 and the effect of the novel drug Semapimod on intestinal inflammation in an experimental model of NEC in neonatal rats. Methods: Newborn rats were subjected to hypoxia and fed a conventional formula by gavage (FFH), or were breast-fed (BF). Rats were sacrificed on day 4, and the distal ileum was harvested for morphological studies and Western blot analysis. Results: FFH newborn rats but not BF controls developed intestinal inflammation similar to the histological changes observed in human NEC. We found that the expression of HMGB1 and its receptor RAGE as well as that of other apoptosis/inflammation-related proteins (Bad, Bax, iNOS, COX-2) was upregulated in the ileal mucosa of FFH newborn rats compared to BF animals. Administration of the drug semapimod inhibited the upregulation of those proteins and partially protected the animals against the formula feeding and hypoxia-induced intestinal injury. Elevated levels of HMGB1 were also found in ileal samples from infants undergoing intestinal resection for acute NEC. Conclusions: Our results implicate HMGB1 and RAGE as important mediators of enterocyte cell death and hypoxia-induced injury in NEC, and support the hypothesis that inhibitors such as semapimod might play a therapeutic role in chronic intestinal inflammation characterized by this animal model.