Objectives Serum calcium levels (sCa) were reported to be associated with risk of cardiovascular diseases. The aim of this study was to analyse the association between sCa and long-term mortality in patients with acute coronary syndrome (ACS). Design A retrospective observational cohort study. Setting Single-centre study with participants recruited from the local area. Participants A total of consecutive 13 772 patients with ACS were included in this analysis. Patients were divided based on their sCa profile (<= 2.1 mmol/L, 2.1-2.2 mmol/L, 2.2-2.3 mmol/L, 2.3-2.4 mmol/L, 2.4-2.5 mmol/L,>2.5 mmol/L) and followed up for a median of 2.96 years (IQR 1.01-4.07). Primary outcome Long-term all-cause mortality. Results During a median follow-up period of 2.96 years, patients with sCa <= 2.1 mmol/L had the highest cumulative incidences of all-cause mortality (16.7%), whereas those with sCa 2.4-2.5 mmol/L had the lowest cumulative incidences of all-cause mortality (3.5%). After adjusting for potentially confounding variables, the Cox analysis revealed that compared with the reference group (sCa 2.4-2.5 mmol/L), all the other groups had higher mortality except for the sCa 2.3-2.4 mmol/L group (HR, 1.32, 95% CI 0.93 to 1.87). Restricted cubic splines showed that the relationship between sCa and all-cause mortality seemed to be U shaped. The optimal sCa cut-off point, 2.35 mmol/L, was determined based on the shape of restricted cubic splines. Conclusions Altered serum calcium homeostasis at admission independently predicts all-cause mortality in patients with ACS. In addition, a U-shaped relationship between sCa and all-cause mortality exists, and maintaining sCa at approximately 2.35 mmol/L may minimise the risk of mortality.