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Background: Previous studies found minimal evidence and raised controversy about the link between hemoglobin and 28-day mortality in sepsis patients. As a result, the purpose of this study was to examine the association between hemoglobin and 28-day death in sepsis patients by analyzing the Medical Intensive Care IV (MIMIC-IV) database from 2008 to 2019 at an advanced medical center in Boston, Massachusetts. Methods: We extracted 34,916 sepsis patients from the MIMIC-IV retrospective cohort database, using hemoglobin as the exposure variable and 28-day death as the outcome variable, and after adjusting for confounders (demographic indicators, Charlson co-morbidity index, SOFA score, vital signs, medication use status (glucocorticoids, vasoactive drugs, antibiotics, and immunoglobulins, etc.)), we investigated the independent effects of hemoglobin and 28-day risk of death by binary logistic regression as well as two-piecewise linear model, respectively. Results: Hemoglobin levels and 28-day mortality were shown to be non-linearly related.The inflection points were 104 g/L and 128 g/L, respectively. When HGB levels were between 41 and 104 g/L, there was a 10% decrease in the risk of 28-day mortality (OR: 0.90; 95% CI: 0.87 to 0.94, p-value = 0.0001). However, in the range of 104–128 g/L, we did not observe a significant association between hemoglobin and 28-day mortality (OR: 1.17; 95% CI: 1.00 to 1.35, P value = 0.0586). When HGB was in the range of 128–207 g/L, there was a 7% increase in the risk of 28-day mortality for every 1 unit increase in HGB (OR: 1.07; 95% CI: 1.01 to 1.15, P value = 0.0424). Conclusion: In patients with sepsis, baseline hemoglobin was related to a U-shaped risk of 28-day death. When HGB was in the range of 12.8–20.7 g/dL, there was a 7% increase in the risk of 28-day mortality for every 1 unit increase in HGB. [ABSTRACT FROM AUTHOR]