Objective  To investigate the effects of lactadherin (MFG-E8) on the repair of intestinal injury and expression of Toll-like receptor 4 (TLR4) of neonatal rats with necrotizing enterocolitis (NEC). Methods  NEC model was established by a combination of aggressive gavage with formula milk, hypoxia, and cold stress. A total of 30 one-day-old newborn rats were randomly divided into the breast milk group (n=10), NEC model group (N group, n=10), and NEC combined with recombinant human MFG-E8 (rhMFG-E8) group (N+M group, n=10). Based on the NEC model, rats of the N+M group were administrated with 100 μg of rhMFG-E8 daily. Rats were sacrificed after being fed for 96 h. Intestinal morphology was observed. The intestinal ileocecum tissue was collected and histopathological examination and transmission electron microscope (TEM) examination were conducted. TLR4 expression of intestinal ileocecum was detected by real-time PCR and Western blotting. Results  The rhMFG-E8 was capable of improving the intestinal appearance of rats with NEC and significantly decreasing the pathological score and incidence of NEC (P<0.05). The expression of TLR4 of the N+M group was significantly lower than that of the NEC model group (P<0.05). Typical apoptotic cells were not observed in intestinal ultra microstructure of the N+M group. Conclusion  Administration of rhMFG-E8 can significantly decrease the expression of TLR4 and apoptosis of intestinal epithelial cells, maintain the integrity of intestinal barrier, and reduce the incidence of NEC of neonatal rats.