Fluid shear stress modulates endothelial cell signalling e.g. by activating protein kinases such as Akt and AMPK. The forkhead box O (FoxO) transcription factors are known substrates of Akt. When phosphorylated, FoxO proteins translocate from the nucleus to the cytoplasm, where they are degraded. Constitutively expressed FoxO1a expression induces expression of various genes, such as angiopoietin-2 (Ang-2), which is postulated to destabilize the vascular wall. We assessed the influence of shear stress on FoxO1a phosphorylation and translocation in HUVECs. Shear stress elicited activation of AMPK and phosphorylation of FoxO1a leading to its translocation and degradation. Overexpression of a dominant negative mutant of AMPK abrogated the shear stress-mediated FoxO1a phosphorylation, while inhibition of Akt was without effect indicating that flow-dependent FoxO1a phosphorylation is mediated in an AMPK-dependent manner. One consequence of the shear stress-induced activation of AMPK and subsequent inactivation of FoxO1a was the downregulation of Ang-2 protein level. Expression of a dominant negative AMPK-mutant led to increased protein levels of FoxO1a and Ang-2, while the expression of a constitutively active AMPK-mutant attenuated them. Our data indicate that shear stress elicits FoxO1a phosphorylation and its translocation, which is AMPK-dependent, but Akt-independent.