Aim: Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a fundamental role in the initiation and development of atherosclerosis. Atorvastatin is known to exert pleiotropic effects on cardiovascular system. This study was designed to examine the effect of atorvastatin on homocysteine (Hcy)-induced activation of ER stress and the potential mechanisms regarding AMP-activated protein kinase (AMPK). Methods and results: Apolipoprotein E-deficient (apoE−/–) mice were administrated with methionine or atorvastatin and sacrificed 2 months later for plasma tests and immunohistochemical analysis. To further study the mechanisms, human umbilical vein endothelial cells (HUVECs) were incubated with various concentrations of Hcy for 1 h, or 500 μmol/L Hcy for 1–24 h. Furthermore, we challenged HUVECs with Hcy in the presence or absence of atorvastatin, 5-amino-4-imidazolecarboxamide riboside-l-beta-D-ribofuranoside (AICAR), an AMPK agonist, and AMPK-DN that expressed a dominant-negative mutant of AMPK. Expression levels of ER stress markers were measured by real-time PCR and Western blot analysis. Our data revealed that atorvastatin prevented Hcy-induced ER stress in the aortic roots of hyperhomocysteinemic mice. In vitro study showed atorvastatin suppressed Hcy-induced ER stress in HUVECs as well. AICAR is found to have the same effect as that of atorvastatin, which could be antagonized by AMPK-DN. Conclusions: Atorvastatin inhibits Hcy-induced ER stress both in vitro and in vivo. The protective effect of atorvastatin against Hcy-induced vascular injury is mediated by AMPK activation.