Objectives: Manganese is one of the essential trace elements that are required by the human body. Klotho protein is a classic anti-aging marker. The association between the levels of serum manganese and serum klotho in individuals between the ages of 40-80 in the United States remains unclear. Methods: Data for this cross-sectional study was obtained from the National Health and Nutrition Examination Survey (NHANES 2011-2016) in the United States. We performed multiple linear regression analyses to investigate the association between the levels of serum manganese and serum klotho. Furthermore, we performed a fitted smoothing curve according to a restricted cubic spline (RCS). Stratification and subgroup analyses were performed for further verification of the results. Results: Weighted multivariate linear regression analysis showed that serum manganese levels were independently and positively associated with serum klotho levels (β = 6.30, 95% confidence interval: 3.30-9.40). Kruskal-Wallis test showed that participants with higher manganese quartiles had higher serum klotho levels (Q1: 808.54 ± 256.39pg/mL; Q2: 854.56 ± 266.13pg/mL; Q3: 865.13 ± 300.60pg/mL; and Q4: 871.72 ± 338.85pg/mL, p < 0.001). The RCS curve indicated that the association between the levels of serum manganese and serum klotho was non-linear. Furthermore, a significantly positive association was found between serum manganese and serum klotho levels in the majority of subgroups. Conclusion: A non-linear and positive association was found between the levels of serum manganese and serum klotho in individuals aged 40-80 in the United States according to the NHANES (2011-2016).