The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (Leucine-rich repeat-containing-G-protein coupled-receptor-5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, as crypt ER stress and apoptosis occurred in TLR4ΔIEC-OVER mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4ΔIEC mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (Protein-kinase related-PKR-like ER-kinase), CHOP (C/EBP-homologous protein) and MyD88 (Myeloid-differentiation primary response-gene-88), but not ATF6 (activating-transcription factor-6), XBP1 (X-box-binding protein-1), or TRIF(TIR-domain-containing adapter-inducing interferon-β). Human and mouse NEC showed high crypt ER stress and apoptosis, while genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intra-gastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis, and suggest that increased ER stress within the premature bowel predisposes to NEC development.