BackgroundSepsis remains a complex condition with incomplete understanding of its pathogenesis. Further research is needed to identify prognostic factors, risk stratification tools, and effective diagnostic and therapeutic targets.MethodsThree GEO datasets (GSE54514, GSE65682, and GSE95233) were used to explore the potential role of mitochondria-related genes (MiRGs) in sepsis. WGCNA and two machine learning algorithms (RF and LASSO) were used to identify the feature of MiRGs. Consensus clustering was subsequently carried out to determine the molecular subtypes for sepsis. CIBERSORT algorithm was conducted to assess the immune cell infiltration of samples. A nomogram was also established to evaluate the diagnostic ability of feature biomarkers via “rms” package.ResultsThree different expressed MiRGs (DE-MiRGs) were identified as sepsis biomarkers. A significant difference in the immune microenvironment landscape was observed between healthy controls and sepsis patients. Among the DE-MiRGs,NDUFB3was selected to be a potential therapeutic target and its significant elevated expression level was confirmed in sepsis usingin vitroexperiments and confocal microscopy, indicating its significant contribution to the mitochondrial quality imbalance in the LPS-simulated sepsis model.ConclusionBy digging the role of these pivotal genes in immune cell infiltration, we gained a better understanding of the molecular immune mechanism in sepsis and identified potential intervention and treatment strategies.