Background/Aims: The death of endothelial cells may play a critical role in the development of various vascular diseases, including atherosclerosis. While free fatty acids (FFAs) may stimulate endothelial apoptosis, the molecular and cellular mechanisms of this effect have not been studied intensively. To elucidate the mechanisms involved in FFA-induced endothelial cell apoptosis, we investigated the effect of different pharmacological inhibitors on palmitate-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Interestingly, lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, showed a strong protective effect. Methods and Results: To examine the involvement of GSK-3β in palmitate-induced HUVEC apoptosis, its dephosphorylation at Ser<sup>9</sup> and enzymatic activation in response to palmitate treatment were monitored by immunoblotting and in vitro kinase assays, respectively. GSK-3β was dephosphorylated and its enzymatic activity increased in palmitate-treated HUVECs. In addition, pretreatment with other GSK-3β inhibitors, e.g. SB216763 or TDZD-8, as well as adenoviral transduction with a catalytically inactive GSK-3β had significant protective effects against palmitate-induced HUVEC apoptosis. Conclusion: These results demonstrate that the GSK-3β signalling pathway is involved in palmitate-induced HUVEC apoptosis.