Background: Oxidative stress and inflammation may contribute to the disruption of the protective gut barrier through various mechanisms; mitochondrial dysfunction resulting from inflammatory and oxidative injury may potentially be a significant source of apoptosis during necrotizing enterocolitis (NEC). Tumor necrosis factor (TNF)α is thought to generate reactive oxygen species (ROS) and activate the apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase (JNK)/p38 pathway. Hence, the focus of our study was to examine the effects of TNFα/ROs on mitochondrial function, ASK1-JNK/p38 cascade activation in intestinal epithelial cells during NEC.Results: We found (a) abundant tissue TNFα and ASK1 expression throughout all layers of the intestine in neonates with NEC, suggesting that TNFα/ASK1 may be a potential source (indicators) of intestinal injury in neonates with NEC; (b) TNFα-induced rapid and transient activation of JNK/p38 apoptotic signaling in all cell lines suggests that this may be an important molecular characteristic of NEC; (c) TNFα-induced rapid and transient ROs production in RIe-1 cells indicates that mitochondria are the predominant source of ROS, demonstrated by significantly attenuated response in mitochondrial DNA-depleted (RIE-1-ρ°) intestinal epithelial cells; (d) further studies with mitochondria-targeted antioxidant PBN supported our hypothesis that effective mitochondrial ROS trapping is protective against TNFα/ROs-induced intestinal epithelial cell injury; (e) TNFα induces significant mitochondrial dysfunction in intestinal epithelial cells, resulting in increased production of mtROS, drop in mitochondrial membrane potential (MMP) and decreased oxygen consumption; (f) although the significance of mitochondrial autophagy in NEC has not been unequivocally shown, our studies provide a strong preliminary indication that TNFα/ROs-induced mitochondrial autophagy may play a role in NeC, and this process is a late phenomenon.Methods: Paraffin-embedded intestinal sections from neonates with NEC and non-inflammatory condition of the gastrointestinal tract undergoing bowel resections were analyzed for TNFα and ASK1 expression. Rat (RIE-1) and mitochondrial DNA-depleted (RIE-1-ρ°) intestinal epithelial cells were used to determine the effects of TNFα on mitochondrial function.Conclusions: Our findings suggest that TNFα induces significant mitochondrial dysfunction and activation of mitochondrial apoptotic responses, leading to intestinal epithelial cell apoptosis during NeC. Therapies directed against mitochondria/ROS may provide important therapeutic options, as well as ameliorate intestinal epithelial cell apoptosis during NeC.