Rhynchosia minima root, a folk herbal medicine in southern China, is used to relieve itch and swelling. In this study, we examined the anti-inflammatory property of an ethanol fraction (EEF6) from R. minima root on lipopolysaccharide (LPS) induced RAW 264.7 cells, as well as its underlying mechanism. The compounds composition of EEF6 was determined by high-performance liquid chromatography-mass spectrometry. The result showed that five flavonoids compounds, 2',4',5,7-Tetrahydroxyisoflavone, Genistein-8-C-glucopyranoside, Tricin, Genistein, and Daidzein, were identified in EEF6. In addition, EEF6 exhibited potent anti-inflammatory ability against LPS stimulated RAW 264.7 cells via MAPK/NF-κB signaling pathways by decreasing the secretion of nitric oxide (NO), interleukin (IL)-6, TNF-α, and monocyte chemotactic protein (MCP)-1, inhibiting the translocation of p65 from cytoplasm to nucleus, and suppressing the phosphorylation of ERK, JNK, and p38. These results indicated that EEF6 could be a promising ingredient for inflammation management.