The prevalence of arterial stiffness increases with age, whereas the level of the aging-suppressor protein klotho decreases with age. The objective of this study is to assess whether haplodeficiency of klotho gene causes arterial stiffness and to investigate the underlying mechanism. Pulse wave velocity, a direct measure of arterial stiffness, was increased significantly in klotho heterozygous (klotho^+/−) mice versus their age-matched wild-type (WT) littermates, suggesting that haplodeficiency of klotho causes arterial stiffening. Notably, plasma aldosterone levels were elevated significantly in klotho^+/− mice. Treatment with eplerenone (6 mg/kg per day IP), an aldosterone receptor blocker, abolished klotho deficiency–induced arterial stiffening in klotho^+/− mice. Klotho deficiency was associated with increased collagen and decreased elastin contents in the media of aortas. In addition, arterial matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-β1 expression and myofibroblast differentiation were increased in klotho^+/− mice. These klotho deficiency–related changes can be blocked by eplerenone. Protein expression of scleraxis, a transcription factor for collagen synthesis, and LC3-II/LC3-I, an index of autophagy, were upregulated in aortas of klotho^+/− mice, which can be abolished by eplerenone. In cultured mouse aortic smooth muscle cells, aldosterone increased collagen-1 expression that can be completely eliminated by small interfering RNA knockdown of scleraxis. Interestingly, aldosterone decreased elastin levels in smooth muscle cells, which can be abolished by small interfering RNA knockdown of Beclin-1, an autophagy-related gene. In conclusion, this study demonstrated for the first time that klotho deficiency–induced arterial stiffening may involve aldosterone-mediated upregulation of scleraxis and induction of autophagy, which led to increased collagen-1 expression and decreased elastin levels, respectively.