Soybean, which contains soyasaponins and isoflavones as representative constituents, exhibits anti-inflammatory and antioxidant effects. To understand the anti-inflammatory effects of soyasaponins, we isolated soyasaponin I, a major constituent of soybean, and investigated the inhibitory effects on inflammatory markers in LPS-stimulated mouse peritoneal macrophages and 3,4,5-trinitrobenzenosulfonic acid (TNBS)-induced colitic mice. Soyasaponin I, which exhibited lipid peroxidation-inhibitory effects in vitro, inhibited the production of proinflammatory cytokines (TNF-α and IL-1β), inflammatory mediators (NO and PGE2), and inflammatory enzymes (COX-2 and iNOS) in LPS-stimulated peritoneal macrophages. Soyasaponin I also suppressed the phosphorylation of IκB-α and the nuclear translocation of NF-κB. However, these soyasaponins barely inhibited mitogen-activated protein kinases. Oral administration of soyasaponin I (10 and 20 mg/kg) to TNBS-treated colitic mice significantly reduced inflammatory markers, colon length, myeloperoxidase, lipid peroxide (malondialdehyde and 4-hydroxy-2-nonenal), proinflammatory cytokines and NF-κB activation in the colon, as well as increased glutathione content, superoxide dismutase, and catalase activity. Based on these findings, soyasaponin I may attenuate colitis by inhibiting the NF-κB pathway.