Changes in the functions of the intestinal barrier occur in parallel with the development of sepsis. The protection by Bifidobacterium strains (BB, BL, BB12, and BLBB) was evaluated in mice injected with lipopolysaccharide (LPS). The results revealed an increase in the ratio of ileal villus length to crypt depth in the BLBB group compared with that in the LPS group, as were the number of IgA+ plasma, CD4+/CD8+ T, and dendritic cells. The levels of diamine oxidase (DAO) and d-lactic acid in the serum were lessened in the BLBB group after LPS injection compared with that in the LPS group. In addition, the BLBB group exhibited an increased expression level of tight junction proteins (zonula occludens-1, occludin, and claudin-1), mucin (MUC2) mRNA, reduced NFκB/MAPK signaling pathways, and decreased expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α). The BLBB group enriched the relative abundance of Muribaculaceae, Lachnospiraceae_NK4A136_group, Clostridia_Ucg-014, and Alistipes, resulting in an increase in strains producing short-chain fatty acids. Furthermore, the BLBB group leads to higher levels of deoxycholic acid and biosynthesized linoleate. This study suggested that the BLBB group could enhance the capacity of the intestinal barrier and intestinal mucosal immunity, reduce intestinal inflammation, and improve the composition of gut microbiota. Bifidobacterium bifidum E3 combined with Bifidobacterium longum subsp. infantis E4 may thus serve as a probiotic against the intestinal injury caused by LPS.