The application of β-amyloid (Aβ) is cytotoxic to endothelial cells, promotes vasoconstriction and impairs nitric oxide (NO) generation or action. However, there is no information on the effect of intracellular Aβ on endothelial cell biology, although recent studies indicate that neuronal Aβ drives Alzheimer’s disease pathogenesis. Since the serine–threonine kinase Akt is crucial to both neuronal and endothelial cell survival as well as eNOS activation, we investigated the effects of Aβ expression on Akt-signaling in cultured endothelial cells. Virally-encoded Aβ42 was proapoptotic and inhibitory to Akt phosphorylation in human umbilical vein endothelial cells (HUVECs). Toxicity was characterized by mitochondrial dysfunction, DNA condensation and activation of caspase-3. Substrates downstream of Akt action, GSK-3β and eNOS, are underphosphorylated in the presence of Aβ. Constitutive activation of Akt reversed Aβ-induced toxicity and stimulated caspase-3 activity, suggesting that inhibition of Akt signaling is functionally significant. These Aβ effects were mediated, in part, through the derepression of GSK-3β activation and correlated with reductions in NO production. We conclude that intracellular production of Aβ42 is cytotoxic to endothelial cells and that disruption of the Akt/GSK-3β cell signaling pathway is involved.