Sepsis is a complex disease characterized by an inflammatory response and tissue hypoxia. Hypoxia-inducible factor-1α (HIF-1α) expression level is regulated by hypoxia and inflammation. This study aimed to explore the correlation between HIF-1α expression level and sepsis by bioinformatics analysis and clinical investigation.Bioinformatics tools were used to identify differentially expressed genes (DEGs) between sepsis and non-sepsis groups using the Gene Expression Omnibus (GEO) dataset. A clinical investigation was carried out to validate HIF-1α protein level in 54 non-septic patients and 173 septic patients who were followed up for 28 days.Bioinformatics analysis revealed that HIF-1α mRNA level was significantly different between septic and non-septic patients (P < 0.05). Consistent with the study hypothesis, higher HIF-1α levels in plasma were found in septic patients compared with those in non-septic patients. The diagnostic accuracy for sepsis, as quantified by the area under the curve, was 0.926 (0.885-0.968) for HIF-1α expression level combined with oxygen saturation to fraction of inspired oxygen (SpO2/ FiO2), white blood cell (WBC) and blood urea nitrogen (BUN). The HIF-1α expression level was also significantly correlated with the severity of the disease. The results of the restricted cubic splines (RCS) model indicated a U-shaped relationship between HIF-1α expression level and intensive care unit (ICU) mortality. Univariate and multivariate linear regression analyses indicated that septic patients with the elevated HIF-1α expression levels had shorter length of ICU stay versus those with the lower HIF-1α expression levels.HIF-1α expression level can be used for diagnosing disease, assessing severity, and predicting length of ICU stay in septic patients.Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American College of Sports Medicine.