Background <p>Neutrophil extracellular traps (NETs) have been demonstrated to play an important role in acute myocardial infarction (AMI), but their specific mechanisms in the development of AMI have not been investigated.</p> Methods <p>The AMI datasets were screened from the GEO database. The hub genes in differentially expressed NET-related genes (DE-NRGs) were screened using bioinformatics and machine learning algorithms, and the nomogram model was constructed based on this. Functional enrichment analysis clarified the biological function and signaling pathways of NETs-mediated AMI. Immune infiltration analysis was performed on the AMI datasets. Finally, the AMI model was constructed using Wistar rats, evaluated by echocardiography and HE staining, and hub genes expression was verified by real-time quantitative PCR (RT-qPCR) and western blot.</p> Results <p>Based on the bioinformatics analysis, 23 DE-NRGs were obtained. The hub genes were screened by machine learning algorithms for NFIL3, MMP9 and S100A8. Immune infiltration analysis demonstrated the relative abundance of immune cells in the sample and their expression levels in hub genes. The experimental results showed that LVEF and FS were significantly reduced in the AMI group, cardiac function was impaired, and there were disturbances in the arrangement of cardiomyocytes and interstitial edema with inflammatory infiltration. NFIL3, MMP9 and S100A8 were significantly elevated in the AMI group compared to the Sham group.</p> Conclusion <p>This study identified the hub genes of NETs-mediated AMI and constructed a diagnostic model through bioinformatics and experimental verification, which provided a new idea for the molecular mechanisms of AMI development and treatment.</p>