This paper was aimed to observe the anti-atheroslerosis effect of paeonol (Pae) on the activation of PI3K/AKT-NF-kappaB and the proliferation activity of rat vasular endothelial cells induced by lipopolysaccharide (LPS) and co-cultured with smooth muscle cells. Primary rat vascular endothelial cells (VECs) and rat vascular smooth cells (VSMCs) were cultured by predigesting and adhering tissue blocks. The VEC-VSMC co-culture model was established by Transwell chamber. LPS (100 mugL ⁻, 7 h) was used to induce VEC injury. MTT assay were used to determine the VEC proliferation activity. Western blot was used to detect PI3K/AKT and NF-kappaB's signaling pathways related protein expressions. The concentration of LPS-induced VECs injury was 100 mugL ⁻, and the time was 7 h. After the intervention on the above cell model for 24 h, paeonol (15, 30, 60 mumolL ⁻) could effectively inhibit LPS-induced VECs injury, block PI3K/AKT-NF-kappaB signal transduction pathway thereby significantly affecting the proliferation of LPS-induced VECs co-cultured with SMCs. The anti-atherosclerosis mechanism of paeonol may be related to the reducing the inhibitory effect of the signaling pathway associated proteins of VEC PI3K/AKT and NF-kappaB, thereby increasing the VEC livability under co-culture.