The relationships of Klotho levels with cognition and dementia are poorly understood. This study aimed to investigate the association between Klotho levels and cognitive function and to determine causality between Klotho and dementia using Mendelian randomization (MR). Based on data from the National Health and Nutrition Survey (NHANES) 2011-2014, this study consisted of 1875 older adults aged 60-79 years. Cognitive function was assessed by the digit symbol substitution test (DSST). We performed weighted multivariable-adjusted linear regression to assess the association between Klotho concentrations and cognitive function. Then, 2-sample MR was conducted to assess the causal relationship between Klotho and dementia. The inverse variance weighted (IVW) method was used as the primary analysis. We observed a positive association between serum Klotho concentrations and the results of the Digit Symbol Substitution test (DSST) (T2: beta 2.16, 95% CI: 0.30-4.01, P = 0.03, T3: beta 2.48, 95% CI: 0.38-4.57, P = 0.02) after adjusting for the covariates. Moreover, there was also a potential nonlinear relationship between Klotho and DSST. The IVW method showed that genetically predicted high Klotho levels were not significantly associate with any type of dementia, including Alzheimer's disease (OR = 1.03, 95% CI: 0.96-1.10, P = 0.46), vascular dementia (OR = 1.04, 95% CI: 0.87-1.25, P = 0.66), frontotemporal dementia (OR = 0.73, 95% CI: 0.47-1.14, P = 0.16), or dementia with Lewy bodies (OR = 1.03, 95% CI: 0.87-1.23, P = 0.73). In the cross-sectional observational study, Klotho and cognitive function were significantly correlated; however, findings from MR studies did not indicate a causal relationship between Klotho and dementia.