Lipopolysaccharides (LPS), a main component of the Gram-negative bacterial cell wall, can damage the epithelial wall barrier and induce chronic intestinal inflammation. The purpose of this study is to evaluate whether the novel L. rhamnosus could alleviate intestinal inflammation and damage induced by LPS and explore the possible underlying molecular mechanism. L. rhamnosus JL-1 was selected from five L. rhamnosus strains due to its strong adherence capacity to Caco-2 cells (92.89%) and it could survive in simulated gastrointestinal juices. Whole genome sequencing analysis showed that there were no translocation and inversion regions in the genome of L. rhamnosus JL-1 compared with L. rhamnosus GG. Comparative genomic analysis showed that there were encoding genes related to adhesion, acid resistance and bile salt resistance in the genome of L. rhamnosus JL-1. Both in vitro and in vivo experiments indicated that LPS challenge inhibited the mRNA and protein expression of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). However, the mRNA and protein expressions of pro-inflammatory cytokines were inhibited by pre-treatment with L. rhamnosus JL-1 in a dose-dependent manner. The result of histopathology analysis of ileum showed that oral administration of L. rhamnosus JL-1 reduced pathological damage induced by LPS. Furthermore, it was revealed that L. rhamnosus JL-1 could inhibit the mRNA and protein expressions of TLR4 and NF-κB. These results strongly suggested that L. rhamnosus JL-1 relieved LPS-induced intestinal inflammation by inhibiting the TLR4/NF-κB signaling pathway. To sum up, L. rhamnosus JL-1 has a potential probiotic function and plays an important role in preventing LPS-induced intestinal inflammation and damage.