Intestinal barrier functions are altered during the development of sepsis. Cathelicidin antimicrobial peptides, such as LL-37 and mCRAMP, can protect animals against intestinal barrier dysfunction. Cathelicidin-BF (C-BF), a new cathelicidin peptide purified from the venom of the snake Bungarus fasciatus, has been shown to have both antimicrobial and anti-inflammatory properties. This study investigated whether C-BF pretreatment could protect the intestinal barrier against dysfunction in a mouse model of endotoxemia, induced by intraperitoneal injection of LPS (10mg/kg). Mice were treated with low or high dose C-BF before treatment with LPS, and samples were collected 5h after LPS treatment. C-BF reduced LPS induced intestinal histological damage and gut permeability to 4 KD Fluorescein-isothiocyanate-conjugated dextran. Pretreatment with C-BF prevented LPS induced intestinal tight junction disruption and epithelial cell apoptosis. Moreover, C-BF down regulated the expression and secretion of TNF-α, a process involving the NF-κB signaling pathway. C-BF also reduced LPS induced TNF-α expression through the NF-κB signaling pathway in mouse RAW 264.7 macrophages. These findings indicate that C-BF can prevent gut barrier dysfunction induced by LPS, suggesting that C-BF may be used to develop a prophylactic agent for intestinal injury in endotoxemia.