Inflammation is the primary defense system in response to external stimuli such as allergic reactions, infections, wounds and chemical irritants. When acute inflammation becomes chronic, it develops to severe diseases including cancer, lupus, fibromyalgia and rheumatoid arthritis. Steroids and nonsteroidal anti-inflammatory drugs have been used as therapeutic agents, but these drugs have a wide range of side effects like heartburn, stomach pain or ulcer, headache, and tiredness. It is necessary to develop anti-inflammatory drugs with less side effects and better efficacy. As a result of our ongoing search for a new natural anti-inflammatory compound from traditional folk medicine, It was found that the methanol extract obtained from the root bark of Robinia pseudoacacia L. inhibited NO and ROS production in the LPS-induced inflammation of RAW 264.7 cell line. R. pseudoacacia (Fabaceae) is distributed in North America, Europe and Asia. The flower and root have been used for hemostasis, arthritis and hypertension. Flavonoids, stilbenes and triterpenes have been isolated from this plant. Previous studies on this plant have reported the anti-inflammatory activity of leaf and stem bark, and anti-oxidant activity of flower. There was no prior study on the biological effect of the root bark. Therefore, this study was accomplished to elucidate the biological activity and the bioactive compounds of the root bark. First, the methanol extract of the root bark was fractionated with methylene chloride, ethyl acetate, butanol, and water, depending on the solvent polarity. Among them, methylene chloride fraction showed the highest inhibitory activity against the LPS-induced inflammation. Seven compounds (1-7) were isolated from this fraction through silica gel and Sephadex LH-20 column chromatography, recrystallization, and prep-HPLC. Based on the spectroscopic data including 1H-NMR, 13C-NMR, EI or ESI-MS data, the chemical structures were determined to be medicarpin (1), vestitol (2), indole 3-carboxaldehyde (3), 3-acetylindole (4), liquiritigenin (5), 4(1H)-quinolone (6) and 8-methoxyononin (7). Among the compounds, medicarpin (1), vestitol (2), 3-acetylindole (4) and liquiritigenin (5) reduced the levels of NO and ROS in a dose-dependent manner. This is the first study to show the anti-inflammatory activity of the root bark of R. pseudoacacia, and it is suggested that the root bark, the methylene chloride fraction, and the compounds (1, 2, 4, and 5) could be useful natural materials for the treatment of inflammatory diseases.