Background: Although drug trials with niacin and cholesteryl ester transfer protein inhibitors that substantially increase high-density lipoprotein-cholesterol (HDL-C) failed to reduce the risk of coronary heart disease, HDL protection of the cardiovascular system cannot be easily denied. Hence, it may be HDL subfractions that are responsible for the long-held and consistent cardioprotective association of HDL. Arterial stiffness has been increasingly recognized as a strong predictor of subclinical vascular disease, atherosclerotic disease, and cardiovascular mortality. As the association of HDL subfractions and arterial stiffness is not well characterized, we aimed to determine the relations between these two entities in a community-based longitudinal Chinese population sample. Methods: We evaluated the associations of plasma HDL2-C and HDL3-C subfractions with arterial stiffness measured using carotid-femoral pulse wave velocity (cf-PWV) and then multivariate logistic regression in 1447 subjects (mean age 61.3 years) from a community-based population in Beijing, China. Results: After a median follow-up of 4.8 years, Pearson's correlation analysis revealed that HDL3-C was negatively associated with follow-up cf-PWV (r = -0.114; P = 0.001), and there was no correlation between HDL2-C and follow-up cf-PWV (r = -0.045; P = 0.181). In the multivariate logistic regression analysis, each standard deviation (SD) increase in HDL3-C was associated with a 1.490-increased likelihood of the presence of follow-up cf-PWV [odds ratio (per SD increase in HDL3-C) 1.490; 95% confidence interval 1.021-1.470; P = 0.039), whereas there was no relation between HDL2-C and follow-up cf-PWV. Conclusions: HDL3-C subfractions were significantly and inversely associated with arterial stiffness, suggesting that HDL subfractions are likely more important than HDL-C in preventing cardiovascular disease.