Fructus Aurantii Immaturus and Radix Paeoniae Alba Powder (FPP) is a popular Chinese herbal prescription. The combination of Fructus Aurantii Immaturus and Radix Paeoniae Alba has been used to treat gastrointestinal disorders for hundreds of years. To our interest, this combination shows a bilateral effect on gastrointestinal peristalsis. Our present study was focused on the bilateral role of this combination on the gastrointestinal tract. The effective constituents and mechanisms were explored. Six monomer constituents from Radix Paeoniae Alba and Fructus Aurantii Immaturus were screened by intestinal transit assay. The bilateral roles of three effective constituents were authenticated by gastric emptying assay, and the combination of three constituents showed a bilateral effect. Then, the mediating receptors and the role of NO and NF- kappaB p65 were examined to determine the mechanism involved. The overall results suggest that the major effective constituents of this combination are synephrine, hesperidin and paeoniflorin. Synephrine inhibits the gastrointestinal movement, while hesperidin stimulates it. Paeoniflorin shows different effects on intestinal and gastric activity. The effect of synephrine relies on the alpha-adrenergic receptor, and the effect of hesperidin is mediated via the H1 histamine receptor. The regulation of hesperidin and synephrine on NF- kappaB p65 translocation and NO production through the alpha-receptor and the H1 receptor, respectively, is involved in the bilateral effect of the Fructus Aurantii Immaturus-Radix Paeoniae Alba combination.