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Background Bronchopulmonary dysplasia (BPD) is a chronic lung disease that occurs in preterm infants and lacks effective treatment. We aim to reveal the relationship between amniotic fluid (AF) peptides and lung development by analyzing the differences in the composition of AF peptides at different gestational periods, thus providing a new means of prevention and treatment for BPD. Methods Based on the stages of lung development, we collected AF by amniocentesis in two different gestational periods, using the 25th week of pregnancy as the cut-off. We conducted a peptide omics analysis of these AF samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Additionally, we verified the regulatory effects of hyperoxia and the peptide COL5A2 on BPD-related cells [(mouse lung epithelial (MLE-12) cells] by 5-Ethynyl-2'-deoxyuridine (EdU) staining, JC-1 staining, flow cytometry, and reactive oxygen species (ROS) assay. Results There were 131 differentially expressed peptides, including 85 up-regulated and 46 down-regulated [fold change (FC) ≥1.2 or ≤1/1.2, P < 0.05], in the ≥25 weeks’ gestation group compared to the <25 weeks’ gestation group. Further bioinformatics analysis revealed that the precursor proteins of the differentially expressed peptides between these two groups were involved in the regulation of the developmental process, anatomical structure development, and other biological processes, suggesting that these differential peptides may play a key role in lung development. We found peptide COL5A2 with the sequence GPPGEPGPPG and verified the regulatory effects of COL5A2 on the proliferation, apoptosis, cell viability, and ROS levels of MLE-12 cells by cell assays. Conclusions In this study, peptidomic studies using AF from different gestational periods revealed that peptides in AF may be involved in lung development. They could be used in the future to assist in the postnatal development of preterm infants and provide new therapeutic prospects for BPD.