Background <p>Sepsis is a critical condition involving dysregulated immune and metabolic responses. Ferroptosis has been implicated in its pathogenesis. However, the precise molecular mechanisms governing ferroptosis in sepsis remain unclear.</p> Methods <p>Multiple transcriptomic datasets related to sepsis were comprehensively analyzed to screen potential hub genes. Differential expression profiling, weighted gene co-expression network construction, and three distinct machine learning approaches were applied in combination. To further elucidate the cellular distribution of these genes, single-cell RNA sequencing data were analyzed, and a corresponding miRNA–mRNA interaction network was established. The biological significance of the identified candidates was subsequently verified through in vitro functional experiments.</p> Results <p>Two core genes were identified. CSTA was significantly upregulated in sepsis and positively correlated with neutrophil infiltration and M1 macrophage polarization. DDX24 was markedly downregulated and positively associated with CD8⁺ T cell and NK cell function. Both genes demonstrated strong diagnostic performance (AUC &gt; 0.87) in validation cohorts. In vitro modulation of these genes revealed that CSTA knockdown or DDX24 overexpression enhanced cell viability and reversed dysregulation of ferroptosis-related proteins, including upregulation of GPX4 and SLC7A11 and downregulation of ACSL4.</p> Conclusions <p>CSTA and DDX24 are central regulators of ferroptosis in sepsis, exhibiting diagnostic and therapeutic potential.</p>