The aim of this study is to explore the protective mechanism of human umbilical cord mesenchymal stem cell exosomes (hUCMSC-Exo) on acute lung injury (ALI) in neonatal rats, and to provide a new method for the treatment of neonatal respiratory distress syndrome (NARDS), This study constructed an animal model of ALI based on neonatal rats. After treatment with hUCMSC-Exo, it was found that the mortality rate of neonatal rats was significantly improved, and the trend of weight loss in neonatal rats was also alleviated. Lung tissue samples were collected from each group of neonatal rats to extract total protein, RNA, and homogenate. Western blot was used to detect the protein expression levels of p-NF-κB, IL-6, Occludin, Clu-din 5, β-Catenin, and Cyclin D1; the mRNA expression levels of TNF-α, IL-1β, IL-6, IL-4, IL-10 and IL-13 were detected by RT-qPCR; enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of TNF-α, IL-6, and IL-10 in lung tissue. The results of pathological sections of lung tissue showed that the infiltration of inflammatory cells, thickening of alveolar septa, pulmonary hemorrhage, and edema were all reduced after treatment with exosomes. The research results indicate that LPS can effectively induce pulmonary inflammation in neonatal rats, leading to acute lung injury, the treatment with exosomes alleviated the degree of ALI. The results of this study can provide new strategies for the treatment of NARDS.