This study was conducted to isolate and identify the anti-inflammatory active peptide in black soybean treated by high hydrostatic pressure after germination. The anti-inflammatory activity was examined using RAW 264.7 macrophages. Black soybean was germinated for 4 days and subjected to 150 MPa for 24 h, followed by preparation of protein extracts. The extracts were sequentially filtered using membranes with a molecular weight cutoff of 30, 10, and 3 kDa. A strong inhibitory effect was observed with the 3–10 kDa fraction. The active peptide from this fraction was purified by gel permeation column chromatography and first and second semipreparative HPLC. After isolation and purification of the active peptide, secretion of inflammatory markers (nitric oxide, TNF-α, IL-1β, and IL-6) by LPS-stimulated RAW 264.7 macrophages was measured. The purified anti-inflammatory peptide was analyzed by UV, LC–MS, 1 H and 13C NMR, COSY, HMBC, and HSQC and was identified as a tripeptide.