The Klotho gene, identified as an 'aging suppressor' gene, encodes a single-pass transmembrane protein. The extracellular domain of Klotho is cleaved and released in the blood stream, where it may function as a vasculoprotective hormone. Carotid artery intima-media thickness (CIMT), flow-mediated dilation (FMD) of the brachial artery and epicardial fat thickness (EFT) have been reported as early predictors of atherosclerosis. We aimed to investigate the relationship between serum Klotho levels and early atherosclerotic predictors, including EFT, FMD and CIMT in healthy adults. Fifty healthy volunteers were enrolled in this study, consisting of 21 males and 29 females with median age of 32 years. They were free of known risk factors for cardiovascular diseases. Serum Klotho levels were determined by the ELISA method. The study population was divided into two groups (n = 25 for each) according to the median serum Klotho level (459.4 pg/mL): higher Klotho (HK) group (613.6 pg/mL; ranges of 501.2-772.6 pg/mL) and lower Klotho (LK) group (338.7 pg/mL; ranges of 278.8-430.3 pg/mL). EFT was measured by transthoracic echocardiography, and CIMT and FMD were measured with standard procedures. The LK group showed lower values of FMD (p = 0.012) and larger values of EFT (p = 0.01) and CIMT (p < 0.001), compared to the HK group. Thus, the low serum Klotho levels were associated with increased EFT and CIMT and with the decreased FMD in the study population. We propose that the lower serum Klotho level is a newly identified predictor of atherosclerosis.