Chlorella is a unicellular green alga, which contains a variety of nutrients including amino acids, dietary fibers, n-3 unsaturated fatty acid, vitamins, and minerals. We previously demonstrated that Chlorella-derived multicomponent supplementation decreases arterial stiffness in young men. However, mechanisms underlying the reduction in arterial stiffness by Chlorella-derived supplementation and the effect in middle-aged and older individuals have remained unexplored. This study tested our hypothesis that Chlorella-derived supplementation improves arterial stiffness via an increase in nitric oxide (NO, a endothelium-derived relaxing factor) production in middle-aged and older individuals. Thirty-two subjects between 45 and 75 years of age assigned to placebo and Chlorella groups in a double-blinded manner and took respective tablets for 4 weeks. Before and after the supplementations, brachial-ankle pulse wave velocity (baPWV, an index of arterial stiffness) and plasma nitrite/nitrate (NOx, end product of NO) concentration were measured. There was no difference in baPWV between before and after the placebo intake, but baPWV decreased after the Chlorella supplementation. Changes in baPWV with the Chlorella supplementation were correlated with those in plasma NOx concentration. We concluded that Chlorella-derived multicomponent supplementation decreases arterial stiffness in middle-aged and older individuals. It may be associated with increase in NO production by vascular endothelium.