Low levels of the Klotho protein are associated with accelerated tissue aging, including arterial stiffness, in patients with cardiovascular and renal diseases. However, this association has not been examined in a diverse cohort. We aimed to investigate the association between serum Klotho protein levels and pulse pressure, as an indicator of arterial stiffness, in a cohort representative of the US population.We used the National Health and Nutrition Examination Survey (NHANES) from 2007 to 2016. Association between pulse pressure and Klotho levels as a continuous variable and in quartiles were examined using survey weight-adjusted linear regression models. Multivariable models were adjusted for age, gender, race, BMI, hypertension, diabetes, smoking, alcohol use, total cholesterol, and estimated GFR.Of the 13,362 participants, 3954(29.6%) were > 65 years, 5792(43%) were Caucasian, and 6773(50.7%) had hypertension. Mean(SD) Klotho was 0.85(0.31) ng/mL and pulse pressure was 55.8(18.5) mmHg. In unadjusted and adjusted models, each ng/mL increase in Klotho was associated with a 3.88mmHg (95%CI = -5.19,-2.57; P < 0.001) and 1.63mmHg (95%CI = -3.01,-0.24; P = 0.02) decrease in pulse pressure, respectively. Similarly, participants in the highest quartile of Klotho had lower pulse pressure than those in the lowest quartile (-3.05mmHg; 95%CI = -4.05,-2.05; P < 0.001), and this difference remained significant in adjusted models (-1.10mmHg; 95%CI = -2.20,-0.01; P = 0.05).In this large diverse cohort, we found an inverse and independent association between serum Klotho levels and pulse pressure suggesting that Klotho is associated with arterial stiffness. The mechanisms underlying this association need further study.