Objective The purpose of this study was to investigate the possible intervention targets and inhibitory effects of melatonin (MT) on Acute lung injury (ALI). Methods The main targets of MT and the disease targets of ALI were obtained through database screening. The common targets of MT and ALI were analyzed by Venn diagram. The network model of “drug-disease” was built by Cytoscape 3.8.2. Protein interactions were analyzed by STRING and PPI networks were constructed. At the same time, an animal model of ALI was established and MT treatment was performed. The changes of lung histopathology were observed. The pulmonary function levels of Ri-area, Re-area and Cldyn-area in each group were detected by tracheotomy and intubation. Results 196 MT targets, 3948 ALI targets and 118 drug-disease co-targets were obtained. EGFR, HIF1A and ERBB2 were the key core sites of MT targeting ALI disease. In the GO enrichment analysis, a total of 1690 biological processes, 90 cellular components and 176 molecular functions were involved. As for KEGG pathway enrichment screening, a total of 157 biological pathways related to this were found. The results of in vivo experiments showed that MT therapeutic intervention had obvious inhibitory effect on ALI. Conclusion MT may inhibit the onset and progression of ALI through multiple targets.