Based on both experimental and clinical data, tamoxifen has been proposed to have cardiovascular benefits, although the mechanism(s) contributing to that protective effect are still poorly understood. In vitro experiments demonstrated that tamoxifen elicits its transcriptional effect through estrogen receptor (ER) alpha, but other targets can participate in its actions. However, although tamoxifen selectively activates the activating function (AF)-1 of ERalpha, we recently showed that this ERalpha subfunction is dispensable for the atheroprotective action of 17beta-estradiol (E2), the main ligand of ERs. The goal of the present work is to determine to which extent ERalpha and its AF-1 mediate the vasculoprotective action of tamoxifen. Our data confirm that tamoxifen exerts an atheroprotective action on low density lipoprotein receptor (LDL-r(-/-)) female mice, but, in contrast to E2, it fails to accelerate reendothelialization after carotid electric injury. Tamoxifen and E2 elicit differences in gene expression profiles in the mouse aorta. Finally, the atheroprotective action of tamoxifen is abrogated in ERalpha(-/-)LDL-r(-/-) mice and in LDL-r(-/-) mice selectively deficient in ERalphaAF-1 (ERalphaAF-1(0/0)LDL-r(-/-)). Our results demonstrate, for the first time to our knowledge, that tamoxifen mediates its actions in vivo through the selective activation of ERalphaAF-1, which is sufficient to prevent atheroma, but not to accelerate endothelial healing.