Necrotizing enterocolitis (NEC) is a devastating disease that predominantly affects premature neonates. The mortality associated with NEC has not changed appreciably over the past several decades. The underlying etiology of NEC remains elusive, although bacterial colonization of the gut, formula feeding, and perinatal stress have been implicated as putative risk factors. The disease is characterized by massive epithelial destruction, which results in gut barrier failure. The exact molecular and cellular mechanisms involved in this complex disease are poorly understood. Recent studies have provided significant insight into our understanding of the pathogenesis of NEC. Endogenous mediators such as prostanoids, cyclooxygenases, and nitric oxide may play a role in the development of gut barrier failure. Understanding the structural architecture of the gut barrier and the cellular mechanisms that are responsible for gut epithelial damage could lead to the development of novel diagnostic, prophylactic and therapeutic strategies in NEC.