A novel oligosaccharide showed that protection against LPS-induced Caco-2 cells was purified from the mycelium of Hericium erinaceus (HE). WEP-1 is mainly composed of neutral monosaccharides with molecular weight of 4,010 Da and of mannose, glucose, and galactose in a molar ratio of 1.2:16.9:1. The structure of WEP-1 includes α-D-Glc (1 → 3) and β-D-Gal (1 → 3) as the backbone with β-D-Glc (1 → 3) as branches attached to the C-4 position and β-D-Man as a terminal residue. The oligosaccharide reduced acetic acid-induced colonic mucosa injury in rats. It also showed significant protection against LPS-induced Caco-2 cells via the TLR4/NF-κB pathway. PRACTICAL APPLICATIONS: In the study, the oligosaccharide from HE has the potential to be developed into functional foods or medicines for the treatment of intestinal diseases. The protection against LPS-induced Caco-2 cells via the TLR4/NF-κB pathway may be a key target for the pharmacological activity of HE.