Telomere length, a marker of biological aging, has been associated with cardiovascular disease (CVD). Increased arterial stiffness, an indicator of arterial aging, predicts adverse CVD outcomes. However, the relationship between telomere length and arterial stiffness is less well studied. Here we examined the cross-sectional association between leukocyte telomere length (LTL) and arterial stiffness in 2,165 American Indians in the Strong Heart Family Study (SHFS). LTL was measured by qPCR. Arterial stiffness was assessed by stiffness index β. The association between LTL and arterial stiffness was assessed by generalized estimating equation model, adjusting for sociodemographics (age, sex, education level), study site, metabolic factors (fasting glucose, lipids, systolic blood pressure, and kidney function), lifestyle (BMI, smoking, drinking, and physical activity), and prevalent CVD. Results showed that longer LTL was significantly associated with a decreased arterial stiffness (β=-0.070, P=0.007). This association did not attenuate after further adjustment for hsCRP (β=-0.071, P=0.005) or excluding participants with overt CVD (β=-0.068, P=0.012), diabetes (β=-0.070, P=0.005), or chronic kidney disease (β=-0.090, P=0.001). In summary, shorter LTL was significantly associated with an increased arterial stiffness, independent of known risk factors. This finding may shed light on the potential role of biological aging in arterial aging in American Indians.