The aqueous extract of Diaphragma Juglandis Fructus (AED), a by-product of Juglans regia L., represents a promising food-derived functional ingredient with potential benefits for intestinal health. This study evaluated the anti-colitis effects of AED and explored its underlying mechanisms using LPS-stimulated RAW264.7 macrophages and a DSS-induced colitis mouse model. In DSS-induced colitis in mice, AED at 10 μg/mL suppressed pro-inflammatory cytokine production and inhibited JAK1/STAT3 signaling. In DSS-induced colitis in mice, AED at 600 mg/kg for 7 days mitigated DSS-induced colonic injury, restored tight junction proteins, and improved epithelial barrier integrity. Integrated transcriptomic and metabolomic analyses identified AED-associated alterations in arginine-polyamine and taurine-hypotaurine metabolism, while network pharmacology and molecular docking suggested angiotensin-converting enzyme (ACE) and von Willebrand factor (VWF) as candidate functional targets for further investigation. Collectively, these findings indicate that AED exerts anti-colitis effects in association with coordinated changes in inflammatory signaling, metabolic pathways, and barrier-related markers, supporting its potential as a food-derived functional ingredient candidate for ulcerative colitis management.