The aim of the research was to investigate the anti-inflammatory mechanism of Pulsatillae Decoction (PD), the levels of interleukin (IL)-6, IL-8, E-selectin, and thromboxane B(2) (TXB(2)) secreted by cultured rat intestinal microvascular endothelial cells (RIMECs) were determined after treatment with its active ingredients, namely anemoside B4, anemonin, berberine, jatrorrhizine, palmatine, aesculin, and esculetin. RIMECs were challenged with 1 microg/mL lipopolysaccharide (LPS) for 3 h, and then treated with each of the seven ingredients at three concentrations (1, 5 and 10 microg/mL) for 24 h. The results revealed that anemonin, aesculin and esculetin inhibited the production of IL-6, aesculin and esculetin inhibited the secretion of IL-8, anemoside B4, berberine and jatrorrhizine downregulated E-selectin expression, anemonin, berberine, jatrorrhizine and palmatine decreased the content of TXB(2). All these changes were significant. Taken together, the data suggest that all seven active ingredients of PD can effectively reduce inflammatory response, thus relieving intestinal dysfunction via multiple pathways.