We investigated the effects of Achillea millefolium extract in vitro on the growth of primary rat vascular smooth muscle cells (VSMCs) as well as the potential involvement of estrogen receptors (ERs) in this process. In addition, the ability of A. millefolium extract to modulate the NF-κB pathway was tested in human umbilical vein endothelial cells (HUVECs). The fingerprinting of the extract was carried out by HPLC-DAD and LC-MS(n) and main constituents were flavonoids (10%) and dicaffeolylquinic acid derivatives (12%). The extract enhanced VSMC growth at least in part by acting through ERs and impaired NF-κB signaling in HUVECs. The various compounds may act with different mode of actions thus contributing to the final effect of the extract. Our findings support some of the traditional uses of A. millefolium, and suggest potential modes of action as related to its effects on vascular inflammation. Therefore, A. millefolium may induce novel potential actions in the cardiovascular system.