BACKGROUND: Necrotizing enterocolitis (NEC) is a critical gastrointestinal disease in preterm infants, for which no specific treatment is established. We previously demonstrated that thrombin-preconditioned mesenchymal stromal cell-derived extracellular vesicles (thMSC-EVs) enhance protection against other neonatal tissue injuries. Therefore, this study aimed to evaluate the therapeutic potential of thMSC-EVs in modified in vitro, in vivo, and organoid models of NEC. METHODS: In vitro, the effects of thMSC-EVs and naïveMSC-EVs were compared in hyperosmotic, ischemic, and hypothermic (HIT)-stressed IEC-6 cells and LPS-treated peritoneal macrophages. In vivo, NEC was induced in P4 mouse pups by three cycles of formula feeding, oral LPS administration, hypoxia, and hypothermia, followed by overnight dam care. 2 × 10 RESULTS: In vitro, compared with naïveMSC-EVs, thMSC-EVs significantly improved cellular viability in HIT-induced IEC-6 cells and reduced pro-inflammatory (IL-1α, IL-1β, TNF-α) but increased anti-inflammatory (TGF-b) cytokine levels in LPS-treated peritoneal macrophages. In vivo, thMSC-EVs significantly attenuated clinical symptoms, reduced intestinal damage, and retained intestinal stem cell markers, showing more significant localization in NEC-induced intestines than in healthy intestines. In NEC mouse-derived organoids, thMSC-EVs significantly increased OLFM4 and claudin-4 expression and reduced stress-related markers such as sucrase-isomaltase, defensin, and chromogranin A. Proteomic analysis revealed that thMSC-EVs were greater enriched in anti-apoptotic, anti-inflammatory, cell adhesion, and Wnt signaling pathways than naïveMSC-EVs. CONCLUSION: thMSC-EVs improved cellular viability, reduced apoptosis, attenuated inflammation, and upregulated key intestinal stem cell markers, collectively suggesting their tissue-protective effects and highlighting their potential as a treatment for NEC.