Combined antiretroviral therapy (cART) has significantly improved the survival rate in HIV-infected individuals, but it contributes to the development of various metabolic complications. Klotho is a novel antiaging gene that encodes a protein with pleiotropic functions, including an emerging role in cardiovascular disease (CVD). The protective effect of higher plasma klotho levels against CVD was recently observed in non-HIV-infected adults. We aimed to assess whether plasma-secreted α-klotho is associated with subclinical carotid atherosclerosis in HIV-infected patients receiving cART. We prospectively examined the association of circulating plasma α-klotho in 120 HIV-infected patients who had received cART for ≥6 months with intima-media thickness (IMT) in the carotid artery and other metabolic variables. The subclinical carotid atherosclerosis was defined as an increased mean IMT level of ≥75th percentile for the matched age, sex, and race and/or the presence of carotid plaque. Thirty-four (28.3%) of 120 had subclinical carotid atherosclerosis. The higher plasma levels of α-klotho had protective effect against subclinical carotid atherosclerosis (OR 0.006, p=0.034) in multivariate regression analysis. Plasma α-klotho levels had a significantly negative correlation with fasting glucose levels (r=-0.216, p=0.018) and mean IMT (r=-0.258, p=0.004) in multiple stepwise regression analyses. The optimal cutoff values of plasma α-klotho levels for the greatest sensitivity and specificity were calculated as 2.83 log10 [pg/ml] (sensitivity, 48.7%; specificity, 90.5%). These results show that plasma klotho levels were inversely associated with subclinical carotid atherosclerosis in HIV-infected patients receiving cART.