Background: Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are severe pulmonary conditions with high morbidity and mortality rates, often triggered by various insults that lead to inflammation and impaired gas exchange. Investigating the molecular mechanisms behind ALI/ARDS is essential for improving diagnostic and therapeutic strategies. Methods: To explore the molecular mechanisms of ALI/ARDS, we utilized bioinformatics, machine learning, and experimental techniques. Transcriptional data from ALI mouse models and control tissues were analyzed, identifying 197 differentially expressed genes (DEGs) with significant upregulation in immune response pathways. Weighted Gene Co-Expression Network Analysis (WGCNA) and machine learning algorithms (SVM-RFE, LASSO, random forest) were used to identify key genes. Additionally, qRT-PCR was employed to assess the expression differences of four core genes related to inflammation. Single-cell sequencing was used to investigate the distribution of Il1r2 in neutrophils, and immunofluorescence co-localization was applied to validate these findings. Results: Four key genes—Cebpd, Hspa12b, Pim1, and Il1r2—were identified as potential biomarkers and therapeutic targets. Il1r2 was notably enriched in neutrophils, highlighting its role in regulating inflammation. Immune cell infiltration analysis revealed increased levels of monocytes, dendritic cells, and neutrophils in ALI samples. qRT-PCR confirmed the expression differences of the four core genes, while single-cell sequencing and immunofluorescence co-localization further validated the distribution of Il1r2 in neutrophils. Conclusion: This study identifies Cebpd, Hspa12b, Pim1, and Il1r2 as key genes in ALI/ARDS, with Il1r2’s expression in neutrophils being particularly significant for understanding inflammation and developing targeted therapies.