Necrotizing enterocolitis (NEC) is characterized by the upregulation of proinflammatory proteins, nitrosative stress, and increased enterocyte apoptosis. We examined the expression and regulation of the Bcl-2/adenovirus EIB 19-kDa-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 and in the liver. In contrast, control rats [breast-fed (BF) without hypoxia] did not develop NEC or elevated BNIP3 expression in these tissues. BNIP3 expression paralleled the histological manifestation of NEC. Supplementation of the formula with L-Nomega-(1-iminoethyl)lysine, 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. Because hypoxia and formation of reactive nitrogen species may promote gut barrier failure, we propose that upregulation of the cell death-related protein BNIP3 is one possible mechanism associated with enterocyte cell death observed in the intestine with NEC.