〔Abstract〕 ObjectiveTo investigate the methylation status of the RUNX family transcription factor 3 (RUNX3) promoter and its mRNA expression in sepsis patients, and to analyze their relationship with the prognosis of sepsis. MethodsDifferentially expressed genes related to sepsis, including RUNX3, were identified from multiple datasets obtained from the gene expression omnibus (GEO) database. The gene expression and methylation sites were validated. A total of 120 patients with sepsis were included. Clinical data were recorded, and blood samples were collected at enrollment. Relative expression levels of RUNX3 in blood samples and promoter methylation status were detected using qPCR and methylation-specific PCR (MSP), respectively. Pearson correlation coefficients were used to analyze the correlation between RUNX3 levels in patient blood and clinical indicators. Kaplan-Meier analysis was performed to plot survival curves, and Cox proportional hazards regression analysis was conducted to identify factors affecting the prognosis of sepsis patients. Results Data set analysis revealed that RUNX3 was a differentially methylated gene associated with the prognosis of sepsis. The mRNA expression level of RUNX3 was lower in the non-survivor group compared to the survivor group ( P < 0.05), and the methylation ratio of RUNX3 was higher in the non-survivor group than in the survivor group ( P < 0.05). In sepsis patients, RUNX3 mRNA expression levels were negatively correlated with Interleukin-6 (IL-6), Procalcitonin (PCT), C-reactive protein (CRP), Acute Physiology and Chronic Health Evaluation (APACHE II) score, and Sequential Organ Failure Assessment（SOFA）score. Kaplan-Meier analysis showed that the 28-day survival rate in the methylated group was lower than that in the unmethylated group ( P < 0.05). Cox regression analysis results indicated that RUNX3 promoter methylation was an independent risk factor for predicting the 28-day prognosis of sepsis patients. ConclusionIn sepsis patients, the mRNA levels of RUNX3 were reduced, and the degree of promoter methylation was higher. RUNX3 promoter methylation was an independent risk factor for the 28-day prognosis of sepsis patients and could serve as a prognostic biomarker for sepsis.