Atheroprotective flow exerts antioxidative and anti-inflammatory effects on vascular endothelial cells (ECs), in part through the induction of Sirtuin 1 (SIRT1), a class III histone deacetylase. The role of Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK)β in flow induction of SIRT1 both in vitro and in vivo was investigated. Pulsatile shear stress mimicking atheroprotective flow increased the level of SIRT1 in cultured ECs by enhancing its stability, and this effect was abolished by inhibition or knockdown of CaMKKβ. Flow-enhanced SIRT1 stability was primarily mediated by CaMKKβ phosphorylation of SIRT1 at Ser-27 and Ser-47, as evidenced by in vitro kinase assay, mass spectrometry, and experiments using loss- or gain-of-function SIRT1 mutants. Flow-induced CaMKKβ phosphorylation of SIRT1 Ser-27 and Ser-47 increased antioxidative and anti-inflammatory capacities. Ablation of CaMKKβ or SIRT1 in mice with an apolipoprotein E-null background showed increased atherosclerosis both in athero-prone and in athero-protective areas. The results suggest that the CaMKKβ-SIRT1 axis in ECs is mechanosensitive, antioxidative, and anti-inflammatory.