Atherosclerosis (AS) is the basis of diabetic macrovascular complications. The plasma low-density lipoprotein (LDL) particles transcytosis across endothelial cells (ECs) and deposition under the endothelium is the initiation step of AS. We previously reported that high glucose inhibits the autophagic degradation of Caveolin-1 and promote LDL transcytosis across ECs, which in turn accelerates atherosclerotic progression. Since Sirt6 is a chromatin-associated protein with deacetylation activity, whether it can regulate Caveolin-1 acetylation and regulating the autophagic degradation of Caveolin-1 remains elusive.Autophagy and histone acetylation were assessed in the umbilical cords of patients with gestational diabetes mellitus (GDM) by immunohistochemistry. An in vitro model of LDL transcytosis was established, and the role of Sirt6 in LDL transcytosis across endothelial cells was clarified. The effect of Sirt6 on the autophagic degradation of Caveolin-1 under hyperglycemic conditions was explored in a streptozotocin (STZ)-induced diabetic AS model established using the ApoE-/- mice.Caveolin-1 and acetylated histone H3 levels were significantly increased, while LC3B and Sirt6 were downregulated in the monolayer of the vascular wall from GDM and type 2 diabetes mellitus (T2DM) patients. Immunoprecipitation assays showed that Sirt6 interacts with Caveolin-1 and specifically mediated its acetylation levels. Immuno-electron microscopy (EM) further indicated that Sirt6 overexpression triggered the autophagic lysosomal degradation of Caveolin-1. ECs-specific overexpression of Sirt6 by adeno-associated viral vector serotype 9 (AAV9) induced autophagy, reduced Caveolin-1 expression, and ameliorated atherosclerotic plaque formation in STZ-induced diabetic ApoE-/- mice.Sirt6-mediated acetylation of Caveolin-1 activates its autophagic degradation and inhibits high glucose-stimulated LDL transcytosis. Thus, the Sirt6/Caveolin-1 autophagic pathway plays a crucial role in diabetic AS, and the overexpression or activation of Sirt6 is a novel therapeutic strategy.