To date, no consensus has been reached regarding the role of klotho in the development of frailty. This study aimed to examine the relationship between serum klotho and physical frailty and to explore potential age, sex, and racial/ethnic differences, using a large, nationally representative sample of middle-aged and older adults in the United States.Cross-sectional study.Participants were 7107 adults aged 45 years or older from the 2007-2008, 2009-2010, 2011-2012, 2013-2014, and 2015-2016 cycles of the National Health and Nutrition Examination Survey (NHANES), a large data set including a series of cross-sectional nationally representative samples in the United States.We assessed the frailty status using the Physical Frailty Phenotype (PFP) and the Frailty Index (FI). Five criteria were used in the PFP, and 34 health items were included to construct the FI as a proportion of accumulated deficits. We used multinomial and binary logistic regression models to examine the association between serum klotho and frailty, adjusted for several covariates.Participants with a higher serum klotho level (>785.5 pg/mL) had a lower prevalence of frailty, defined by either the PFP or the FI, than those with a lower level (≤785.5 pg/mL). After adjustment for all covariates, the higher serum klotho level was associated with a 26% (95% CI 2%-45%) and 17% (95% CI 1%-30%) lower odds of frailty vs robustness when using the PFP and FI, respectively. In the PFP, the association was significantly stronger among participants aged <60 years than those aged ≥60 years (odds ratio: 0.60 vs 0.85; Pinteraction = .03). No effect modification by race/ethnicity on the association was found.Higher serum klotho level relates to lower odds of physical frailty among middle-aged and older adults. Our findings suggest that klotho might be a potential biomarker of frailty, specifically in the middle-aged population. Future research should further investigate the mechanisms underlying this association to determine if lower levels of klotho may serve as a novel risk factor for physical frailty.Copyright © 2023 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.