Sepsis-related acute lung injury (ALI) is a severe and life-threatening complication characterized by excessive inflammation and immune dysfunction. Macrophages play a central role in the pathogenesis of ALI, with dysregulated polarization and impaired phagocytic function contributing to disease progression. This study investigates the role of the Specificity Protein 1 (SP1)/Prohibitin 1 (PHB1) axis in regulating macrophage polarization and function in sepsis-associated ALI. Using a cecal ligation and puncture (CLP)-induced mouse model, combined with single-cell RNA sequencing (scRNA-seq), high-throughput sequencing (HTS), and machine learning (ML) algorithms, we identified SP1 and PHB1 as key regulators of ALI. Functional assays demonstrated that SP1 transcriptionally suppresses PHB1 expression, promoting M1 macrophage polarization while impairing phagocytic function, thereby exacerbating sepsis-induced ALI. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (RT-qPCR), and Western blot analyses further validated these findings. In vivo and in vitro studies confirmed that overexpression of SP1 significantly increased lung inflammation, fibrosis, and apoptosis, whereas PHB1 overexpression reversed these pathological effects. The SP1/PHB1 axis was found to modulate macrophage-mediated immune responses through inflammatory cytokine secretion, mitochondrial dysfunction, and oxidative stress. These findings provide novel insights into the molecular mechanisms underlying sepsis-induced ALI and highlight the potential of targeting the SP1/PHB1 axis for therapeutic intervention. Future research should explore targeted therapies aimed at modulating macrophage polarization and improving immune homeostasis in sepsis-associated ALI.