BACKGROUND: Klotho is a geroprotective protein which has been recognized for its anti-aging properties. Pre-clinical evidence suggested that boosting Klotho might hold therapeutic potential in ageing and disease. Epilepsy is a neurological disorder characterized by its recurrent seizures. The complex interplay between Klotho and epilepsy has not been elucidated. The main objective was to investigate the role of Klotho in epilepsy with combination of observational and mendelian randomization (MR) studies. METHODS: The observational data set comprised 13,766 adults who were aged 20-80 years from the National Health and Nutrition Examination Survey (NHANES) between 2007 and 2016. We used weighted multivariable-adjusted logistic regression models to examine the association between Klotho and epilepsy. We also applied MR to discern if a causal link is present between Klotho and epilepsy. RESULTS: In NHANES study, the incidence of epilepsy tended to decline with an increase of Klotho levels after covariate adjustments. Klotho was identified to have causal effects on epilepsy. MR analyses revealed that higher transformed Klotho (by rank-based inverse normal transformation) levels were correlated with a higher likelihood of developing generalized epilepsy, lesion-negative focal epilepsy, and focal epilepsy, indicating that higher Klotho concentrations were associated with reduced risks of epilepsy. The sensitivity analyses upheld these consistent relationships. CONCLUSIONS: Our research, encompassing comprehensive NHANSE analysis and MR methods, revealed that an increase in Klotho levels was associated with a reduced risk of epilepsy, suggesting that increasing or restoring Klotho might play a protective role and offer new anti-aging therapeutic potential in epilepsy.