Endothelial dysfunction and low-grade inflammation are associated with cardiovascular disease. Arterial stiffening plays an important role in cardiovascular disease and thus may be a mechanism through which endothelial dysfunction and/or low-grade inflammation lead to cardiovascular disease. We investigated the associations between, on the one hand, biomarkers of endothelial dysfunction (soluble endothelial selectin, thrombomodulin and both vascular- and intercellular adhesion molecules 1 and von Willebrand factor) and of low-grade inflammation (C-reactive protein, serum amyloïd A, interleukin 6, interleukin 8, tumour necrosis factor α and soluble intercellular adhesion molecule 1) and, on the other hand, arterial stiffness over a 6-year period, in 293 healthy adults (155 women). Biomarkers were combined into mean Z-scores. Carotid, femoral and brachial arterial stiffness and carotid-femoral pulse wave velocity were determined by ultrasonography. Measurements were obtained when individuals were 36 and 42 years of age. Associations were analysed with generalised estimating equation and adjusted for sex, height and mean arterial pressure. The endothelial dysfunction Z-score was inversely associated with femoral distensibility [β(95%CI)−0.51(−0.95;−0.06)] and compliance coefficients [−0.041(−0.076;−0.006)], but not with carotid or brachial stiffness or carotid-femoral pulse wave velocity. The low-grade inflammation Z-score was inversely associated with femoral distensibility [−0.51(−0.95;−0.07)] and compliance coefficients [−0.050(−0.084;−0.016)], and with carotid distensibility coefficient [−0.91(−1.81;−0.008)], but not with brachial stiffness or carotid-femoral pulse wave velocity. Biomarkers of endothelial dysfunction and low-grade inflammation are associated with greater arterial stiffness. This provides evidence that arterial stiffening may be a mechanism through which endothelial dysfunction and low-grade inflammation lead to cardiovascular disease.