The development and formation of mammalian blood vessels are closely related to the regulation of signal transduction pathways. Klotho/AMPK and YAP/TAZ signaling pathways are closely related to angiogenesis, but the internal relationship between them is not clear. In this study, we found that Klotho heterozygous deletion mice (Klotho+/- mice) had obvious thickening of the renal vascular wall, obvious enlargement of vascular volume, and significant proliferation and pricking of vascular endothelial cells. Western blot showed that the expression levels of total YAP protein, p-YAP protein (Ser127 and Ser397), p-MOB1, MST1, LATS1, and SAV1 in renal vascular endothelial cells were significantly lower in Klotho+/- mice than in wild-type mice. Knockdown of endogenous Klotho in HUVECs accelerated their ability to divide and form vascular branches in the extracellular matrix. Meanwhile, the results of CO-IP western blot showed that the expression of LATS1 and p-LATS1 interacting with AMPK protein decreased significantly, and the ubiquitination level of YAP protein also decreased significantly in vascular endothelial cells of kidney tissue of Klotho+/- mice. Subsequently, continuous overexpression of exogenous Klotho protein in Klotho heterozygous deficient mice effectively reversed the abnormal renal vascular structure by weakening the expression of the YAP signal transduction pathway. Therefore, we confirmed that Klotho and AMPKα proteins were highly expressed in vascular endothelial cells of adult mouse tissues and organs; this resulted in a phosphorylation modification of YAP protein, closed the activity of the YAP/TAZ signal transduction pathway, and inhibited the growth and proliferation of vascular endothelial cells. When Klotho was absent, the phosphorylation modification of YAP protein by AMPKα was inhibited, resulting in the activation of the YAP/TAZ signal transduction pathway and finally inducing the excessive proliferation of vascular endothelial cells.© The author(s).