Probiotics can regulate gut microbes to maintain human health. However, the sensitivity of probiotics to environmental conditions reduces their bioavailability. In contrast, the formation of probiotic biofilm provides a natural physical barrier against external interference. Our previous study established a dynamic culture system of the biofilm-state Bifidobacterium adolescentis Gr19 (B-DC-B. adolescentis Gr19), forming higher density and more structurally stable biofilms, which enhanced its potential probiotic properties in vivo. Thus, the protective effect and mechanism of B-DC-B. adolescentis Gr19 on lipopolysaccharide (LPS)- induced intestinal barrier dysfunction were investigated in this study. The results showed that B-DC-B. adolescentis Gr19 not only had high resistance and adhesion activity, but also improved the intestinal barrier by increasing goblet cells and promoting the expression of tight junction (TJ)-related proteins. Moreover, B-DC-B. adolescentis Gr19 effectively attenuated intestinal barrier injury in Caco-2 cells by improving intestinal permeability and integrity. Remarkably, B-DC-B. adolescentis Gr19 enhanced expression of TJ proteins, restored localization of cytoskeleton and reduced intestinal inflammation by suppressing the Ras homolog family member A/Rho-associated coiled-coil-forming kinases/nuclear factor kappa B/myosin light chain kinase/myosin light chain (RhoA/ ROCK/NF-κB/MLCK/MLC) pathway. Therefore, B -DC-B. adolescentis Gr19 plays a key role in mitigating LPS-induced intestinal barrier dysfunction. Overall, the present study provides a theoretical basis for ameliorating intestinal barrier dysfunction and developing novel functional foods by using biofilm-state probiotics under dynamic culture.