This study explores the functions of exosomes derived from human breast milk (HBM) in vivo and in vitro. HBM-derived exosomes were collected from healthy lactating mothers. In vitro analysis were divided into five groups: (1) a control with no added agents, (2) exosomes added, (3) stimulated with lipopolysaccharide (LPS), (4) pretreated with exosomes and stimulated with LPS, and (5) pretreated with exosome-free HBM and stimulated with LPS. For in vivo analysis, mouse pups were randomly assigned to four groups: (1) a control group of breastfed pups, (2) necrotizing enterocolitis-induced (NEC) pups, (3) pups pretreated with HBM-derived exosomes 6 h before being induced by NEC, and (4) pups pretreated with exosome-free HBM 6 h before NEC induction. Expression of zonula occludens-1 (ZO-1), claudin-1, and occludin were decreased in groups 3 and 5. In the animal model, mice pups in group 3 showed milder intestinal tissue injury than those in group 2 or 4 and had lower levels of the proinflammatory cytokines and higher levels of epithelial tight-junction proteins than groups 2 and 4. HBM-derived exosomes exert beneficial effects in preventing NEC by reducing inflammation and injury to the intestinal epithelium as well as by restoring intestinal tight-junction proteins. HBM-derived exosomes can help protect the epithelial tight-junction proteins ZO-1, claudin, and occludin from inflammatory attack. This study sought (1) to analyze whether there were differences in exosome levels between the human breast milk (HBM) of mothers who had delivered preterm or at term and (2) to investigate whether these exosomes could help sustain the intestinal epithelial tight-junction proteins ZO-1, claudin-1, and occludin in the presence of NEC in vitro and in vivo.