In order to investigate the preventive effect and mechanism of krill oil (KO) on lipopolysaccharide (LPS)-induced intestinal barrier injury, thirty-two male mice were randomly divided into four groups:normal control group (control group), model group (LPS group), KO group and fish oil (FO) group. The mice in control and LPS groups were given olive oil by gavage, while KO and FO groups were fed with 400mg/kg (based on body weight) of KO and FO (diluted in olive oil) orally once a day. After 4 weeks of intervention, the mice were intraperitoneally injected with 10 mg/kg (based on body weight) of sterile normal saline or the same amount of LPS in control group and the other three groups and were sacrificed 6 hours later. The histopathological changes of small intestine among four groups were observed by hematoxylin-eosin staining. The diamine oxidase (DAO) activity in the serumand small intestine of mice were determined. The protein levels of tight junction proteins and inducible nitric oxide synthase (iNOS) were measured by Western blot. The myeloperoxidase (MPO) and nitric oxide (NO) content were also determined. The mRNA expression of inflammatory cytokines was evaluated by quantitative real-time PCR. The protein levels of key factors involved in TLR4/NF-κB signaling pathway were also analyzed. The present findings revealed that KO intervention significantly suppressed the decrease in the ratio of villus length to crypt depth after treated with LPS. KO pretreatment also decreased the serum DAO activity, but increased the intestinal DAO activity in LPS-treated mice. The protein levels of tight junction proteins such as Claudin-1, Occludin and ZO-1 in small intestine were up-regulated by KO pretreatment. In addition, KO also decreased the MPO activity, iNOS protein level, NO content, the mRNA expression of TNF-α, IL-1β and IL-6 as well as the protein levels of key factors involved in TLR4/NF-κB signaling pathway in comparison with those in LPS group. The present study indicates that KO prevents LPS-induced intestinal barrier injury via inhibiting TLR4/NF-κB signaling pathway, and its effect is superior to FO.