Arteriosclerosis caused by aging is recognized to be a crucial risk factor of cardiovascular disease. We recently establishedklothomouse which causes age-related disorders including arteriosclerosis. However, no information on endothelial function ofklothomouse or the physiological role of klotho protein as a circulating factor is available. In this report, we demonstrate that 50% effective dose of aortic relaxation in response to acetylcholine in heterozygousklothomice is significantly greater (4 × 10⁻⁵M) than in wild-type mice (8 × 10⁻⁶M, n = 7, p < 0.05) and that the vasodilator response of arterioles to acetylcholine is significantly attenuated in heterozygous (20% effective dose; 2 × 10⁻⁶M) and homozygousklothomice (>1 × 10⁻⁵M) as compared with wild-type mice (1 × 10⁻⁷M, n = 7, p < 0.05). Nitric oxide metabolites (NO⁻₂and NO⁻₃) in urine are significantly lower in heterozygousklothomice (142 ± 16 nmol/day) than wild-type mice (241 ± 28 nmol/day, n = 13, p < 0.05). Parabiosis between wild-type and heterozygousklothomice results in restoration of endothelial function in heterozygousklothomice. We conclude that the klotho protein protects the cardiovascular system through endothelium-derived NO production by humoral pathways.