Necrotizing enterocolitis (NEC) is the leading cause of neonatal gastrointestinal mortality; effective interventions are lacking with limited understanding of the pathogenesis of NEC. The importance of Toll-like receptor 4 (TLR4) signaling in NEC is well documented; however, the potential mechanisms that regulate enterocyte inflammation and apoptosis remain unclear. The aim of this study was to characterize the role of TLR4-mediated inflammation and apoptosis in the development of NEC and to determine the major apoptotic pathways and regulators in the process. TLR4-deficient C57BL/10ScNJ mice and lentivirus-mediated stable TLR4-silent cell line (IEC-6) were used. NEC was induced by formula gavage, cold, hypoxia, combined with lipopolysaccharide in vivo or lipopolysaccharide stimulation in vitro. Enterocyte apoptosis was evaluated by TUNEL or Annexin analysis. The expression of TLR4, caspase3, caspase8, caspase9, Bip, Bax, Bcl-2, and RIP was detected by Western blot and immunofluorescence. Inflammatory factors such as tumor necrosis factor-α and interleukin-2 were examined by Luminex. Defect of TLR4 led to suppressed enterocytes apoptosis both in vitro and in vivo; the expression of caspase3, caspase8, Bip, and Bax was decreased; and caspase9 and Bcl-2 were increased. NEC severity was attenuated in TLR4-deficient mice compared with wild-type counterparts, and enterocytes apoptosis was correlated with NEC severity. RIP and cytokine level of tumor necrosis factor-α and interleukin-2 were also decreased. TLR4-induced inflammation and apoptosis play a critical role in the pathogenesis of NEC. TLR4 inhibition, combined with extrinsic (caspase8) and/or endoplasmic reticulum stress (Bip) apoptosis signaling blockade could serve as a potential effective treating strategy for NEC.