Background: The link between total cholesterol (TC) and all-cause and specific mortality has not been elucidated. Herein, we aimed to evaluate the effect of TC levels on all-cause, cardiovascular disease (CVD), and cancer mortality.Methods: All data analyzed were obtained from the National Health and Nutrition Examination Survey 1999-2014. The relationship between levels of TC and mortality was determined through Cox proportional hazard regression analysis coupled with multivariable adjustments. Two-piecewise linear regression model and Cox models with penalized splines were applied to explore non-linear and irregular shape relationships. Kaplan-Meier survival curve and subgroup analyses were conducted.Results: The sample studied comprised 17853 men and 18922 women, categorized as 27927 adults aged 18–65 years and 8848 adults more than 65 years old. A total of 4441 deaths were recorded. All-cause, cardiovascular, and cancer mortality showed U-curve associations with nadir at 213 mg/dL, 200 mg/dL, and 218mg/dL after adjusting for confounding variables in the restricted cubic spline analysis. HRs of all-cause, CVD, and cancer mortality was particularly negatively related to TC levels in the lower range <200 mg/dL, especially in the range <120 mg/dL (HR 1.95; 95% CI 1.59, 2.38, HR 1.78; 95% CI 1.16, 2.72, HR 2.18; 95% CI 1.46, 3.25, respectively). The lowest cumulative survival rate was recorded in the lowest TC level group.Conclusions: A U-curve association of TC level with all-cause, cancer, and CVD mortality in the American population was observed, suggesting that too low or too high serum total cholesterol levels might correlate with adverse outcomes.