Background: Serum calcium levels disorder have been reported to be associated with poor prognosis in different diseases. Studies on the association between serum calcium and outcomes of septic patients remained limited. The aim of this study is to investigate the association between serum calcium and 28-day mortality in septic patients. Method: Patients diagnosed with sepsis in the Medical Information Mart for Intensive Care III database were included. Patients were divided into five groups according to the quintiles of serum calcium levels, and their baseline characteristics were compared. Multivariate Cox regression models were used to assess the association between serum calcium and 28-day mortality. Smooth curve fitting and segmented regression models were used to visualize the association between serum calcium levels and 28-day mortality risk. The 28-day survival probability between five groups was analyzed using Kaplan-Meier curves. Results: A total of 3,016 patients with sepsis were enrolled, and the 28-day mortality rate was 35.64%. After adjusting for confounders, compared with the reference quintile (Q4: 9.00–9.50), the lowest serum calcium level quintile (Q1: 5.70–8.20) was independently associated with an increased risk of 28-day mortality (hazard ratio [HR], 2.12; 95% CI, 1.76–2.56). Smooth spline fitting revealed a U-shaped association between serum calcium and 28-day mortality. When serum calcium was 9.0 mg/dL, the 28-day mortality risk increased by 12% per unit increase in serum calcium (HR, 1.12; 95% CI, 1.04–1.20). Conclusion: A U-shaped association was observed between serum calcium levels and 28-day mortality in septic patients. Lower or higher serum calcium levels were associated with increased risk of 28-day mortality in septic patients.