Necrotizing enterocolitis (NEC) is characterized by the upregulation of pro-inflammatory proteins, nitrosative stress, and increased enterocyte apoptosis. We examined the expression and regulation of the Bcl-2/adenovirus EIB 19kD-interacting protein 3 (BNIP3), a pro-apoptotic gene regulated by nitric oxide (NO) in hepatocytes, in NEC. Newborn rats subjected to hypoxia and fed a conventional formula by gavage (FFH) developed NEC and demonstrated elevated expression of BNIP3 mRNA and protein in mucosal scrapings of the ileal samples as well as in the liver. In contrast, control rats (breast-fed without hypoxia) did not develop NEC nor elevated BNIP3 expression in these tissues. BNIP3 expression paralleled the histological manifestation of NEC. Supplementation of the formula with L-NIL, an inducible NO synthase inhibitor, reduced BNIP3 expression in FFH animals to the levels found in BF animals. Both hypoxia and peroxynitrite upregulated BNIP3 protein expression in human intestinal cells. Finally, ileal samples obtained from infants undergoing surgical resection for acute NEC demonstrated higher levels of BNIP3 protein. Since hypoxia and formation of reactive nitrogen species may promote gut barrier failure, we propose that up-regulation of the cell death-related protein BNIP3 is one possible mechanism associated with enterocyte cell death observed in the intestine with necrotizing enterocolitis.