Abstract: OBJECTIVE To study the effects of Liuweidihuang Formula (LWDHF) on the ovariectomized ApoE-/- AS mice and the proliferation and migration of vascular smooth muscle cell (VSMCs) in vitro， and to elucidate the roles of estrogen receptor β (ERβ) and heat shock protein 27 (HSP27) phosphorylation in the context. METHODS The artery injury of ApoE-/- mice was observed by HE staining and the phosphorylation of HSP27 in vessels was detected by immunohistochemistry. VSMC proliferation and cell cycle were examined by MTT assay and flow cytometry， respectively. VSMC migration was observed by wound-healing assay and F-actin staining. The levels of HSP27， pHSP27， cyclin D1， protein phosphatase 2 (PP2A) and ERβ were determined by Western blot analyses. Transfection with ERβ siRNA and ERα shRNA was carried out to investigate their roles in LWDHF effects， respectively. RESULTS LWDHF improved the arterial lesion， inhibited HSP27 phosphorylation， and increased the expression of PP2A and ERβ in ovariectomized ApoE-/- AS mice. In in vitro experiments， angiotensin II (Ang Ⅱ) at 1×10-7 mol/L obviously induced VSMC proliferation and migration， increased the expression of cyclin D1 and cell number in the S phase， but these effects were significantly inhibited by LWDHF (12 μg/mL). Further molecular examinations showed that LWDHF up-regulated PP2A expression through ERβ to inhibit HSP27 phosphorylation， contributing to inhibition of VSMC proliferation and migration. CONCLUSION LWDHF reduced menopause AS in vivo and inhibited proliferation and migration of VSMCs by regulating ERβ-inhibition of HSP27 phosphorylation in vitro. These discoveries provided novel molecular insights into LWDHF as potential therapy for AS.